Methods and compositions for detecting and treating a subset of human patients having an autoimmune disease

ABSTRACT

Provided are monoclonal antibodies, fragments, and derivatives thereof reactive with an epitope of the T cell receptor alpha chain variable region, Vα12.1, on human T lymphocytes. The monoclonal antibodies are reactive with approximately 2% of CD4 +  T lymphocytes and with approximately 5% of CD8 +  T lymphocytes in peripheral blood cells in normal individuals and define a subset of individuals afflicted with an autoimmune disease, especially rheumatoid arthritis, that exhibit increased expression of the Vα12.1 gene on CD8 +   peripheral blood T lymphocytes when compared to normal individuals. Also provided are methods for diagnosing, treating, and monitoring the progression of rheumatoid arthritis in a subject using Vα12.1-specific reagents, including antibodies and nucleic acid probes. Higher levels of assurance in the diagnosis of RA can be made by establishing that the expansion of Vα12.1 gene usage is clonal or oligoclonal and that the Vα12.1 expansion correlates with the occurrence of the MHC allele, HLA-DQw2.

GOVERNMENT RIGHTS IN INVENTION

This invention was made with the support of Government Grant AR39582 andGovernment Fellowship AR0806901 from the National Institutes of Health.The government of the United States of America has certain rights inthis invention.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.07/750,986, filed on Aug. 28, 1991 now abandoned.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to the diagnosis and treatment of a subset ofindividuals afflicted with an autoimmune disease, especially rheumatoidarthritis, using a Vα12.1 T cell receptor-specific-reagent. Expressionof the Vα12.1 gene product on CD8⁺ T lymphocytes is elevated in a subsetof rheumatoid arthritis patients when compared to the expression ofVα12.1 on CD8⁺ lymphocytes of normal, healthy subjects that do not havethe immune abnormality, thereby enabling the diagnosis and treatment ofthis subset of patients.

A higher level of assurance in the diagnosis of rheumatoid arthritis byVα12.1 expansion can be made by determining that the expansion is clonalor oligoclonal and that the expansion correlates with the occurrence ofthe HLA-DQw2 allele in Vα12.1-elevated individuals.

2. Background Art

T cell precursors migrate from the bone marrow to the thymus wherethymocytes begin to mature and express the T cell receptor for antigen.Through positive and negative selection, CD4⁺ T cells generally becomerestricted to major histocompatibility complex (MHC) class II moleculesand CD8⁺ T cells to MHC class I molecules. These CD4⁺ or CD8⁺ T cellsenter the periphery, where, after encountering antigen, they might beinduced to become effector cells or long-lived memory cells.

The recognition of antigen and self-MHC is largely carried out byclonotypic αβ T-cell receptors (TCR), present on the surface ofT-lymphocytes in association with the CD3 complex. Like theimmunoglobulins, TCR α and β chains are assembled by somaticrecombination of discontinuous germline gene segments duringdevelopment. The generation of a highly diverse repertoire of human αβTCRs is accomplished by the recombination of a single V gene segment(selected from a pool of approximately 100 Vαs and 80 Vβs) to anindividual J gene segment (out of approximately 100 Jαs and 13 Jβs).Template-independent nucleotide (N-segment) insertions at the junctions,Dβ usage, and the imprecise joining of the germline gene segmentsfurther increase TCR diversity. Lai et al, Adv. Immunol, 46:1 (1989);Davis et al, Nature, 334:395 (1988). A dominant usage of certain Vβ genefamilies in normal human peripheral T cells has been demonstrated. Choiet al, Proc. Natl. Acad. Sci. USA, 86:8941 (1989). A differential usageof specific α/β genes between CD4⁺ and CD8⁺ peripheral T lymphocytes hasalso been found. Kisielow et al, Nature, 333:742 (1988).

In autoimmune diseases, T cells are believed to act as causative agentsthat incorrectly recognize the host body as foreign. In the autoimmunedisease rheumatoid arthritis (RA), the local site of tissue injury isthe joint. Joint pathology, characterized by inflammation and jointdestruction, is the result of a complex interaction of cellular elements(inflammatory cells, immunocompetent cells and synovial lining cells)and their secreted products (Zvaifler, Am. J. Med., 75:3 (1983)). Thesynovial tissue (pannus) in RA has the appearance of a hypercellularlymphoid organ in which the predominant lymphocytes are T cells, whichmake up 80% of the synovial tissue lymphocytes (Bankhurst et al, Arth.Rheum., 19:555 (1976); Kurosaka, J. Exp. Med., 158:1191 (1983)). Inaddition to the preponderance of T cells in the synovial pannus and anincreased number of these T cells being activated in vivo, evidence fordefects in T cell function and proportions have been described.Importantly, therapeutic measures that alter T cell function, such astotal nodal lymphoid irradiation, significantly improve the clinicaldisease state but are associated with marked toxicity (Kotzin et al, N.Engl. J. Med., 305:969 (1981)).

The relative risk of RA is high in individuals who inherit certain majorhistocompatibility complex genes. HLA DR4 and HLA DR1, for example, arepresent in more than 90% of adult rheumatoid arthritis patients. Whilethe molecular basis for this genetic predisposition is unknown, becausethe major function of the MHC is to present processed antigens to Tlymphocytes, it has been hypothesized that an environmental antigen orinfection initiates an MHC-restricted immune response mediated at leastinitially by T cells in RA.

For many of these reasons, scientific investigators searching for thecausative agent of rheumatoid arthritis have postulated that immunedysregulation associated with the disease may be associated withclonally expanded T cells detectable as a skewing of the peripheral αβ Tcell antigen receptor repertoire. U.S. Pat. No. 4,886,743, for example,describes methods for diagnosing autoimmune diseases, includingrheumatoid arthritis, based upon an expansion of V β gene usage in thetotal population of T lymphocytes in RA patients. PCT InternationalPublication No. 90/06758, which broadly covers monoclonal antibodiesreactive with epitopes on the T cell antigen receptor that can be usedin the diagnosis and treatment of many immune-related diseases,associates rheumatoid arthritis with an increased percentage of T cellswhich express Vδ1, Vβ3, Vβ9, or Vβ10 T cell receptor variable regions ina patient sample. Methods of diagnosing and treating RA patients withmonoclonal antibodies specific for these gene products are described.

Despite the foregoing discoveries, investigators in the field ofautoimmune disease pathology continue to seek correlations between the Tcell antigen receptor and this chronic disease that could form the basisfor future diagnostic and therapeutic modalities.

It is therefore an object of the present invention to provide a strongcorrelation between a variable region of the alpha chain of the T cellantigen receptor and rheumatoid arthritis.

Another object of the invention is to provide diagnostic methods andcompositions for diagnosing and monitoring the progression of rheumatoidarthritis in a distinct subpopulation of RA patients.

A still further object of the invention is to provide therapeuticmethods and compositions for treating a distinct subpopulation ofindividuals with RA.

SUMMARY OF THE INVENTION

These as well as other objects and advantages are achieved in accordancewith the present invention, which provides monoclonal antibodiesreactive with an epitope of the T cell receptor alpha chain variableregion, Vα12.1, on human T lymphocytes. The monoclonal antibodies of theinvention are reactive with approximately 2% of CD4⁺ T lymphocytes andapproximately 5% of CD8⁺ T lymphocytes in peripheral blood cells innormal individuals, as determined, for example, by cytofluorographicanalyses, and define a subset of individuals afflicted with anautoimmune disease, especially rheumatoid arthritis, that exhibitincreased expression of the Vα12.1 gene product on CD8⁺ peripheral bloodT lymphocytes when compared to normal individuals. Also provided arederivatives or fragments of the anti-Vα12.1 monoclonal antibodiesreactive with the Vα12.1 variable region of the alpha chain of a T cellantigen receptor.

In another embodiment, the present invention provides a method fordiagnosing a subset of individuals afflicted with an autoimmune disease,especially rheumatoid arthritis, by detecting an increase in the numberof CD8⁺ T cells in a sample that express the Vα12.1 gene product. Thediagnostic method comprises (A) contacting a suitable sample containingT lymphocytes obtained from a patient suspected of having an autoimmunedisease with a Vα12.1-specific reagent capable of binding to a cellularcomponent of T cells and detecting Vα12.1 gene usage; (B) detecting thebinding of the reagent and determining the number of CD8⁺ T lymphocytesexpressing Vα12.1; and (C) comparing the number of CD8⁺ T lymphocytes asdetermined in step (B) with the number of CD8⁺ T lymphocytes expressingVα12.1 in a baseline sample, for example, a test sample from normalindividuals that do not have the immune system disorder, to determinewhether the number of CD8⁺ T cells expressing the Vα12.1 gene iselevated in the subject suspected of having the disease relative to thenumber of CD8⁺ T cells expressing the sequence in a normal subject. Inone embodiment, the reagent is a monoclonal antibody or derivative orfragment thereof, reactive with an epitope of the Vα12.1 gene product.

A higher level of assurance can be made in the diagnosis of rheumatoidarthritis by establishing that the expansion of Vα12.1 gene usage is aclonal or oligoclonal expansion and by establishing that the Vα12.1expansion correlates with the occurrence of the MHC allele, HLA-DQw2, inthe Vα12.1- elevated patient population.

Diagnostic kits are also provided and comprise in packaged combinationsa reagent, which can be a monoclonal antibody or fragment, capable ofbinding to T cells and detecting the presence of Vα12.1, or a labeledderivative of the reagent.

In another embodiment, rheumatoid arthritis can be treated byadministering a therapeutically effective amount of a Vα12.1-specificreagent alone, or conjugated to a cytotoxic reagent.

The invention also provides for therapeutic compositions comprising themonoclonal antibodies of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fluorescence pattern obtained on the FACSCAN flowcytometer (Becton Dickinson) after reacting cells from the leukemia cellline HPB-MLT, peripheral blood cells from a normal healthy patient, andleukemia cells from the Jurkat leukemia cell line, with the monoclonalantibody 6D6, specifically reactive with the Vα12.1 gene product. Asillustrated by the fluorescent patterns, mAb 6D6 stained HPB-MLT cellsand a minor subpopulation (2-5%) of peripheral blood lymphocytes but notthe Jurkat T leukemia cell line. Staining of Jurkat cells and PBL withthe Vβ 8-specific mAb C305 is shown for comparison. Isotype matched mAbP3 was used as a negative control and mAb T3b (anti-CD3) was used aspositive control.

FIG. 2A illustrates that 6D6 immunoprecipitates the T cell receptor α/βheterodimer. Immunoprecipitations of the TCR complex from cell surface¹²⁵ I radiolabeled HPB-MLT cells were resolved by SDS-PAGE and analyzedby autoradiography. Antibodies were P3 (negative control), Leu4(anti-CD3), βF1 (anti-β framework) and 6D6 (anti-Vα12.1). Undernon-reducing (NR) conditions (lanes 1-4), the αβ TCR complex wasresolved as a 85 kD species by direct immunoprecipitation with mAbs βF1and 6D6 or by co-immunoprecipitation with anti-CD3 mAb Leu 4. Underreducing (R) conditions (lanes 5-8), the TCR α and β subunits werevisualized as 46 kD and 39 kD species, respectively. CD3 subunits arepresent at 20-30kD.

FIG. 2B illustrates that mAb 6D6 recognized a determinant present on theα chain of the HPB-MLT TCR. HPB-MLT cells were metabolically labeledwith ³⁵ (S)-methionine and -cysteine for 4 hours and solubilized in 1%Triton X-100. Immunoprecipitations were carried out with the indicatedantibodies and resolved by SDS page under non-reducing (NR) conditionsand visualized by fluorography. MAbs βF1 (TCR β framework) and 3A2(anti-Vβ5) immunoprecipitated the unpaired β chain (lanes 2 and 3respectively). In contrast, αF1 (anti-framework) and 6D6 antibodies(lanes 4 and 5, respectively) immunoprecipitated the unpaired α chainfrom HPB-MLT lysates. While mAb αF1 immunoprecipitated all of theglycosylated α-chain species identified in HPB-MLT, mAb 6D6immunoprecipitated the mature form of these species.

FIG. 3 illustrates that the Vα12.1 gene segment is rearranged in allcells recognized by mAb 6D6. DNA from 6D6⁺ T-cell clones HD5.A (lane 4)and HD5.B (lane 5), T-cell leukemia cell line HPB-MLT (lane 3), and the6D6⁻ T-cell clone 3A2.D were digested with Bam HI and analyzed bySouthern hybridization using a Vα12.1-specific probe. Germlineconfiguration (GL) for Vα12.1 gene was seen as a 6.3 Kb fragment in theB cell line, SB (lane 1). While both Vα12.1 alleles in 3A2.D T-cellclone were deleted, DNA from HD5.A, HD5.B and HPB-MLT showed distinctVα12.1 rearrangements (R).

FIG. 4A illustrates the Vα12.1 expression in CD4⁺ and CD8⁺ T cells fromadult and newborn peripheral blood lymphocytes, as determined by FACStwo color immunofluorescence analysis. PBMC from a normal healthy adultand a newborn were stained with anti-CD4 (OKT4) or anti-CD8 (OKT8)(green fluorescence, FITC) and anti-Vα12.1 (6D6) (red fluorescence, PE)mAbs as described in Example 2. Lymphocytes were gated on the basis offorward and side scatter profiles (not shown) and analyzed forfluorescence intensity in log scale. Dot plots were divided intoquadrants to represent unstained cells (lower left, quadrant 3), cellsstained with FITC alone (lower right, quadrant 4), cells stained with PEalone (upper left, quadrant 1) and cells that double stained with FITCand PE (upper right, quadrant 2).

FIG. 4B illustrates the Vα12.1 expression in CD4⁺ and CD8⁺ cells from 20normal healthy adults and 10 newborns The expression of Vα12.1 in CD4⁺or CD8⁺ subsets for each individual are connected by a line. The formulaused to calculate the percentage value for 6D6 expression in CD4⁺ orCD8⁺ T cells is set forth in Example 2.

FIG. 5 illustrates the elevated expression of Vα12.1 CD8⁺ T cells in asubset of rheumatoid arthritis patients when compared to T cells fromhealthy individuals. The percentage of mAb 6D6⁺ (Vα12.1 bearing) in CD4and CD8 T cell subsets was determined as in FIG. 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to monoclonal antibodies thatrecognize an epitope of the Vα12.1 variable region of the T cell antigenreceptor and to methods of diagnosing, treating, and monitoring theprogression of rheumatoid arthritis in a subpopulation of RA patientsthat exhibit a clonal or oligoclonal expansion of peripheral blood Tcells expressing Vα12.1 using Vα12.1-specific reagents.

As used herein, the phrase "Vα12.1-specific reagent" means a reagentthat is capable of binding to a cellular component of T lymphocytes anddetecting the T cell receptor Vα12.1 variable region gene or geneproduct thereof. Such reagents include nucleic acid sequences or probesthat can hybridize to nucleic acid sequences within the Vα12.1 geneitself and to antibodies that bind to an epitope of the Vα12.1 geneproduct on T cells for diagnosis and monitoring of the disease processand also include antibodies for treatment of a subpopulation of RApatients exhibiting an expansion of Vα12.1 gene usage.

In the following description, reference will be made to variousmethodologies known to those of skill in the art of immunology, cellbiology, and molecular biology. Publications and other materials settingforth such known methodologies to which reference is made areincorporated herein by reference in their entireties as though set forthin full.

1. Antibodies Of The Invention

The present invention is directed to an antibody or fragment orderivative thereof, specific for an epitope of the Vα12.1 variableregion of the T cell antigen receptor. The antibodies of the inventionare useful in diagnosis and therapy of an autoimmune disease, preferablyrheumatoid arthritis (RA). The term "antibody" is meant to includepolyclonal antibodies, monoclonal antibodies, (mAbs) chimaeric, andhumanized antibodies (see below). Monoclonal antibodies, and chimaericand humanized antibodies, are preferred for diagnosis and therapy,respectively.

A Vα12.1-specific monoclonal antibody of the present invention,designated 6D6, enabled the analysis of the expression of the Vα12.1gene product in peripheral blood T cell subsets of normal, healthy humansubjects by flow cytometry. The results of this analysis revealed thatapproximately 2% of CD4⁺ peripheral blood lymphocytes express the Vα12.1gene product, while about 5% of CD8⁺ peripheral blood lymphocytesexpress Vα12.1. When expression of Vα12.1 was examined in patients withseveral autoimmune diseases, a marked expansion of Vα12.1 in the CD8⁺peripheral blood T cells of a subset of rheumatoid arthritis patientswas detected.

In accordance with the present invention, all of the teachings andprocedures described herein with respect to 6D6 can be used by personsskilled in this area of technology to develop other antibodiesspecifically reactive with an epitope of Vα12.1.

a. Preparation of Monoclonal Antibodies

The monoclonal antibodies of the invention can be prepared by using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the original techniques of Kohler and Milstein, Nature, 265:495-497(1975), modified as described in Brenner et al, J. Immunol., 138:1502(1987), the pertinent portions of which are hereby incorporated byreference and the more recent human B cell hybridoma technique,EBV-hybridoma technique, and trioma techniques well known to personsskilled in the art.

As part of the production of the monoclonal antibodies of the invention,various host animals, including but not limited to rabbits, mice,hamsters, and rats can be immunized by injection with purified T cellantigen receptors or polypeptides or fragments thereof, a recombinant orsynthetic version thereof, or T lymphocytes.

In generating the monoclonal antibodies of the present invention, anycell line that uses the Vα12.1 gene can be used in the immunizationprocedure. An example of human T cell line known to use Vα12.1 is theHPB MLT human leukemia cell line (Sim et al, Nature, 312:771 (December1984)). Whole T cells naturally expressing Vα12.1, such as HPB-MLT, aswell as partially purified T cell receptors can be employed asimmunogens to generate monoclonal antibodies specific to the variableregion of the alpha chain. As an example, partially purified T cellreceptor complexes isolated from the human T cell leukemia cell lineHPB-MLT can be used as the immunogen. Preferably, T cell receptorcomplexes are partially purified by immunoprecipitation using anti-CD3mAb adsorbed to Protein A bearing fixed Staphylococcus aureus bacteria(Pansorbin, Calbiochem, San Diego, Calif.) using the protocol previouslydescribed, J. Immunol., 138:1502 (1987), although any suitable methodfor partially purifying the T cell receptor for antigen from whole Tcells can satisfactorily be employed. Alternative methods of partialpurification will be readily apparent to persons skilled in this area oftechnology.

Whole cells that can be used as the immunogens to produceVα12.1-specific monoclonal antibodies also include recombinanttransfectants that express TCR including Vα12.1. In a particularlypreferred embodiment, human Vα12.1 is transfected into suitable cellsfrom another species such that the human Vα12.1 gene product isexpressed on the cell surface in combination with the T cell receptorfrom another species. As an example, human Vα12.1 is transfected into amurine T-T hybridoma prepared from a murine T cell line, such asBW-1100.129 or BW-1100.125, which are defective in that they fail toexpress the α chain of the T cell receptor for antigen. These cell lineshave been described by White et al in Journal of Immunology,143:1822-1825 (September 1989) and are readily available. Thetransfectants thus prepared are then used in the immunization procedure.This procedure is described by Choi et al, Nature, 346:471 (August1990), the pertinent portions of which are incorporated by reference,and has as an advantage increasing the efficiency of an effective immuneresponse to the human antigen. The cDNA sequence for Vα12.1 is known(Sim et al, Nature, 312:771 (1984)) and can readily be prepared fortransfection by persons skilled in the art for generation of monoclonalantibodies in accordance with the teachings and procedures hereindescribed.

Other Vα12.1 TCR-containing samples that can be used in the immunizationprotocol include peptide sequences that correspond to Vα12.1 andrecombinantly expressed and purified proteins produced from expressionsystems.

Samples of the whole T cells, partially purified T cell receptor proteinor other suitable immunogen can then be injected into a host animal, forexample a mouse and, after a sufficient time, the animal is sacrificedand spleen or other immune cells obtained. The spleen or other immunecells are immortalized by fusing the spleen cells with an immortalizedcell line, generally in the presence of a fusion enhancing reagent, forexample, polyethylene glycol. The resulting cells, which include thefused hybridomas, are then allowed to grow in a selective medium, suchas HAT medium, and the surviving cells are grown in such medium usinglimiting dilution conditions. The cells are grown in a suitablecontainer, e.g., microtiter wells, and the supernatant is screened formonoclonal antibodies having the desired specificity.

In a preferred embodiment, the monoclonal antibodies of the presentinvention are prepared substantially as follows. About 5×10⁶ wholeHPB-MLT cells are injected intraperitoneally into 8 week old Balb/c micein complete Freund's adjuvant. Four to five more intraperitonealimmunizations are conducted at two to three week intervals, also withabout 5×10⁶ HPB-MLT cells in incomplete Freund's. The final boost ismade intravenously into the tail vein, without adjuvant. The mice aresacrificed about three days following the final boost and spleensremoved. The immune spleen cells are then removed and fused with anappropriate myeloma cell line, such as P3X63Ag8.653,in the presence ofpolyethylene glycol in accordance with standard techniques.

Screening procedures that can be used to screen hybridoma cellsexpressing anti-Vα12.1 monoclonal antibody include but are not limitedto (1) enzyme-linked immunoadsorbent assays (ELISA), (2)immunoprecipitation and (3) fluorescent activated cell sorter (FACS)analyses. Many different ELISAS that can be used to screen foranti-Vα12.1 monoclonal antibodies can be envisioned by persons skilledin the art. These include but are not limited to formats comprisingpurified, synthesized or recombinantly expressed Vα12.1 polypeptideattached to a solid phase or formats comprising the use of whole T cellsor cell lysate membrane preparations either attached to the solid phaseor bound to antibodies attached to the solid phase. Samples of hybridomasupernatants would be reacted with either of these two formats, followedby incubation with, for instance, goat-anti-mouse immunoglobulincomplexed to an enzyme-substrate that can be visually identified.

Where the immunogen comprises whole T cells or T cells transfected withhuman Vα12.1-containing TCR, screening is preferably conducted usinghuman HBP-MLT in a FACS analysis.

It is also possible to screen antibodies for their ability toimmunoprecipitate a known Vα12.1 as analyzed by SDS-polyacrylamide gelelectrophoresis or Western blot analysis.

After initial screening, further characterization of the hybridomas forthose that secrete monoclonal antibody specific for the Vα12.1 geneproduct can be accomplished substantially as described in the Examplesherein. Monoclonal antibodies specific for an epitope of Vα12.1 willhave the same TCR chain specificity as 6D6, as determined, for example,by immunoprecipitation using a cell line known to express Vα12.1.Monoclonal antibodies that compete with 6D6 in competition assays wellknown to persons skilled in the art are likely to recognize essentiallythe same epitope as 6D6, while monoclonal antibodies that fail tocompete are likely to recognize a different epitope of the Vα12.1 geneproduct. The specificity of the monoclonal antibody for a Vα12.1sequence can be confirmed by cloning, hybridization and sequencingtechniques, as described in Example 1. Additional confirmatory analyseswhich can be conducted include preclearing and/or cross-blockingtechniques well known to persons skilled in the art.

A molecular clone containing a DNA sequence of an antibody to an epitopeof Vα12.1 can be prepared by known techniques. (See, e.g., Sambrook,Fritsch and Maniatis, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). RecombinantDNA methodology may be used to construct nucleic acid sequences whichencode a monoclonal antibody molecule or antigen binding region thereof.

Once the desired hybridoma has been selected and cloned, the resultantantibody may be produced in one of two major ways. The purest monoclonalantibody is produced by in vitro culturing of the desired hybridoma in asuitable medium for a suitable length of time, followed by the recoveryof the desired antibody from the supernatant. The length of time andmedium are known or can readily be determined. This in vitro techniqueproduces essentially monospecific monoclonal antibody, essentially freefrom other species of anti-human immunoglobulin. However, the in vitromethod may not produce a sufficient quantity or concentration ofantibody for some purposes, since the quantity of antibody generated isonly about 50 μg/ml.

To produce a much larger quantity of monoclonal antibody, the desiredhybridoma may be injected into an animal, such as a mouse. Preferablythe mice are syngeneic or semi-syngeneic to the strain from which themonoclonal-antibody producing hybridomas were obtained. Injection of thehybridoma causes formation of antibody producing tumors after a suitableincubation time, which will result in a high concentration of thedesired antibody (about 5-20 mg/ml) in the ascites of the host animal.

Antibody molecules can be purified by known techniques, e.g. byimmunoabsorption or immunoaffinity chromatography, chromatographicmethods such as high performance liquid chromatography or a combinationthereof.

Following these protocols, any person skilled in this area of technologycan readily isolate hybrid cell lines which secrete a monoclonalantibody exhibiting specificity for an epitope of Vα12.1. Although onlya single hybridoma producing a monoclonal antibody against human Vα12.1antigen is exemplified by way of working example, it is contemplatedthat the present invention encompasses all monoclonal antibodiesexhibiting the characteristics described herein.

For example, it was determined by immunodiffusion that the subjectantibody (6D6) belongs to the subclass IgG₁, which is one of foursubclasses of murine IgG. These subclasses of immunoglobulin G differfrom one another in the so-called "fixed" regions, although an antibodyto a specific antigen will have a so-called "variable" region which isfunctionally identical regardless of which subclass of immunoglobulin Git belongs to. That is, a monoclonal antibody exhibiting thecharacteristic described herein may be of subclass IgG₁, IgG₂ a, IgG₂ b,or IgG₃, or of classes IgM, IgA, or other known Ig classes. Thedifferences among these classes or subclasses will not affect theselectivity of the reaction pattern of the antibody, but may affect thefurther reaction of the antibody with other materials, such as (forexample) complement or anti-mouse antibodies. Although the subjectantibody is specifically IgG₁, it is contemplated that antibodies havingthe patterns of reactivity illustrated herein are included within thesubject invention regardless of the immunoglobulin class or subclass towhich they belong.

Moreover, while the specific example of the novel antibody of thepresent invention is from a murine source, this is not meant to be alimitation. The above antibody and those antibodies having thecharacteristics of the 6D6 antibody, whether from a murine source, othermammalian source including human, rat, or other sources, or combinationsthereof, are included within the scope of this invention, as set forthabove.

b. Preparation of Fragments and Derivatives of Antibodies MolecularFragments and Derivatives

Also included within the scope of the present invention are antibodyfragments and derivatives which comprise at least the functional portionof the antigen binding domain of the anti-Vα12.1 antibody molecule.

Antibody fragments which contain the binding domain of the molecule canbe generated by known techniques. For example, such fragments include,but are not limited to: the F(ab')₂ fragment which can be produced bypepsin digestion of the antibody molecule; the Fab' fragments which canbe generated by reducing the disulfide bridges of the F(ab')₂ fragment,and the Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent. See, e.g., NationalInstitutes of Health, 1 Current Protocols In Immunology, Coligan et al.,ed. §§ 2.8, 2.10 (Wiley Interscience, 1991).

Antibody fragments also include Fv fragments, i.e., antibody products inwhich there are no constant region amino acid residues. Such fragmentscan be produced, for example as described in WO 92/04381 or U.S. Pat.No. 4,642,334.

When antibodies produced in non-human subjects are used therapeuticallyin humans, they are recognized to varying degrees as foreign and animmune response may be generated in the patient. One approach forminimizing or eliminating this problem, which is preferable to generalimmunosuppression, is to produce chimaeric antibody derivatives, i.e.antibody molecules that combine a non-human animal variable region and ahuman constant region. Chimaeric antibody molecules can include, forexample, the antigen binding domain from an antibody of a mouse, rat, orother species, with human constant regions. A variety of approaches formaking chimaeric antibodies have been described and can be used to makechimaeric antibodies containing the immunoglobulin variable region whichrecognize the gene product of Vα-12.1. See, for example, Morrison etal., Proc. Natl. Acad. Sci. U.S.A. 81:6851 (1985); Takeda et al., Nature314:452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al.U.S. Pat. No. 4,816,397; Tanaguchi et al., Eur. Patent Pub. EP171496;Eur. Patent Pub. 0173494; United Kingdom Patent GB 2177096B. Suchchimaeras produce a less marked immune response than non-chimaericantibodies.

For human therapeutic purposes, the Vα12.1-specific monoclonal orchimaeric antibodies can be further humanized by producing humanconstant region chimaeras, in which even parts of the variable regions,especially the conserved or framework regions of the antigen-bindingdomain, are of human origin and only the hypervariable regions are ofnon-human origin. Such altered immunoglobulin molecules may be made byany of several techniques known in the art, (e.g., Teng et al., Proc.Natl. Acad. Sci. U.S.A., 80:7308-7312 (1983); Kozbor et al., ImmunologyToday, 4:7279 (1983); Olsson et al., Meth. Enzymol., 92:3-16 (1982)),and are preferably made according to the teachings of PCT Pub. WO92/06193 or EP 0239400. There are also a number of companies thathumanize antibodies commercially, for example Scotgen Limited, 2 HollyRoad, Twickenham, Middlesex, Great Britain.

These humanized antibodies are preferable for immunotherapy in that theyminimize the effects of an immune response. This in turn leads to alowering of any concomitant immunosuppression and to include increasedlong term effectiveness in, for instance, chronic disease situations orsituations requiring repeated antibody treatments.

Chemical and Biological Derivatives for Diagnosis and Therapy

In addition to molecular antibody fragments and derivatives, antibodyderivatives or immunoconjugates consisting of an antibody molecule orbinding region thereof bound to a label such as a radioisotope,flourescein, enzyme, or other tracer molecule can be made by techniquesknown in the art. Alternatively, the antibody molecule or fragmentthereof can be bound to a therapeutically useful biological or chemicalmolecule targeted to its desired site of action by virtue of theantibody's binding specificity. As one example of such an embodiment, acytotoxic compound can be conjugated to an antibody of the inventionwhich is specific for Vα12.1⁺ lymphocytes which are the causative agentsof an autoimmune disorder. The cytotoxic compound, which can be forexample, a radionucleotide or a toxin, such as diphtheria toxin, inconjugated form is thus targeted to the implicated T lymphocytes.

c. Antibody Isotype

In addition to the properties described supra, the isotype of theanti-Vα12.1-specific antibody is also important. For different clinicalapplications, an antibody of a specific isotype may be preferable to oneof a different isotype. For example, the IgG2a isotype reacts with Fcreceptors on cells of the reticuloendothelial system and is more readilyremoved from the circulation and sequestered in the spleen than otherisotypes. Such an antibody that has reacted with a target cell mayresult in the more efficient removal of the target cell from the site ofactive disease. In addition, some isotypes (such as IgG2a) are moreeffective in antibody dependent cell cytotoxicity reactions than others.In general, antibodies of the IgG isotype are preferable to those of theIgM isotype because they have higher binding affinities.

The desired isotype of an antibody may be selected by screeningpotential antibodies by an ELISA assay designed to select the isotype ofinterest. For example, the solid phase can be coated with goatanti-mouse IgG Fc specific antibodies, if it is desired to select forantibodies having the IgG isotype.

Given an antibody of one isotype, it is also possible to switch theisotype to a different isotype. Many methods for accomplishing thisswitch are known to those skilled in the art. For example, the isotypeswitch can be done by repeatedly selecting for the isotype of interestusing magnetic beads (super paramagnetic iron oxide particles, Biomag©beads purchased from Advanced Magnetics, Inc.) coated with a goatanti-mouse antibody preparation including all isotype classes. Inswitching the isotype from IgG1 to Igg2a, for instance, the IgG2abinding sites on the coated magnetic beads are first blocked with anirrelevant antibody of the IgG2a isotype. All cells producing antibodiesof differing isotypes will then be bound by the beads and removedmagnetically, resulting in an enrichment of cells producing the IgG2aisotype. These cells can then be cloned by limiting dilution, and usingcommercially available anti-isotypic reagents in an ELISA assay, theIgG2a producing clones can be identified. A method which facilitatesselecting for the isotype of interest is exemplified in PCTInternational Publication No. WO 90/06758.

d. Immunoassays

The antibodies of the invention and the fragments and derivativesthereof containing the binding region (e.g., Fab, Fab', F(ab')₂), can beused in various immunoassays Such immunoassays include, but are notlimited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked lmmunosorbentassay), "sandwich" immunoassays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, and immunoelectrophoresis assays,to name but a few.

2. Diagnosing Rheumatoid Arthritis With

Vα12.1-Specific Reagents

The monoclonal antibodies, fragments, and derivatives thereof of thepresent invention, as well as other Vα12.1specific reagents, are usefulfor studying the T cell receptor repertoire in a variety of immunedisorders and are particularly useful in diagnosing and/or monitoringthe disease process in a subset of human patients afflicted withrheumatoid arthritis. Thus, in one embodiment of the inventionrheumatoid arthritis is diagnosed and monitored by detecting thespecific binding of a reagent having specificity for a cellular T cellcomponent that is indicative of the presence and usage of the Vα12.1gene.

a. Antibodies

In a particularly preferred embodiment, the reagent is a monoclonalantibody or fragment or derivative thereof, reactive with an epitope ofthe Vα12.1 gene product on CD8⁺ T lymphocytes in a suitable patientsample. Rheumatoid arthritis is diagnosed by detecting increasedexpression of Vα12.1 on CD8⁺ T cells in the patient sample when comparedto a suitable base line sample, as, for example, a sample from a normalhuman subject that does not have the immune disease.

A suitable sample for diagnosis includes cells from essentially any bodyfluid, including but not limited to peripheral blood, plasma, lymphaticfluid, and synovial fluid, to name just a few. Alternatively a tissuesample, such as synovial membrane, from the site of suspected injury maybe obtained by biopsy and subjected to testing. In a particularlypreferred embodiment of the invention, rheumatoid arthritis is diagnosedby detecting elevated numbers of Vα12.1 positive cells on CD8⁺ Tlymphocytes from peripheral blood relative to the population of Vα12.1positive CD8⁺ T cells in normal individuals.

A diagnostic method in accordance with the present invention involvescontacting the sample containing T cells obtained from a subjectsuspected of having an autoimmune disease, such as rheumatoid arthritis,with a monoclonal antibody or fragment or derivative thereof specificfor an epitope of the Vα12.1 gene product on T cells, detecting thebinding of antibodies to the Vα12.1 gene product on the T cells anddetermining the number of CD8⁺ T cells expressing the Vα12.1 geneproduct. The results are then compared to the number of CD8⁺ Tlymphocytes thus determined as expressing the Vα12.1 gene product withthe number of CD8⁺ T cells expressing the Vα12.1 gene product determinedfor a normal subject. The presence of an increased number of CD8⁺ Tcells expressing the Vα12.1 gene product relative to the number ofVα12.1⁺ CD8⁺ T cells of a normal subject is diagnostic for a subset ofpatients with the disease. In accordance with the present invention, thenumber of CD8⁺ T cells expressing the Vα12.1 gene product can bedetermined either qualitatively or quantitatively.

When used in a diagnostic assay as described, the antibody is typicallylabeled so that its binding with the T cell receptor can be detected.Any suitable label well known to persons skilled in the art, includingbut not limited to fluorescent dyes, radioactive isotopes, enzymes whichcatalyze a reaction producing detectable products, biotin, or metal ionsdetectable by nuclear magnetic resonance can be employed.

Binding of the monoclonal antibodies may be accomplished in vivo or invitro.

In vitro binding may be performed using histological specimens orsubfractions of tissue or fluid, i.e., substantially purified T cells.For preparation of lymphocytes from a sample of peripheral blood for invitro binding, mononuclear cells are separated from the rest of theblood. For example, peripheral blood is anticoagulated with heparin,diluted with an equal volume of phosphate buffered saline and subjectedto density gradient centrifugation, thereby separating the mononuclearcells (primarily lymphocytes) from the rest of the blood. The cells arethen washed in buffer and suspended in buffer with a small amount, e.g.,0.01-1%, preferably about 0.1%, gelatin and a small amount, e.g.,0.01-1%, preferably about 0.1%, sodium azide at 5×10⁶ per ml.Alternatively, separation of the white blood cells can be achieved bywhole blood hemolysis.

Cryostat sections from tissue obtained from biopsy are prepared bycutting the tissue into thin sections, preferably about 6 μm thick, atabout -25° C. and then allowing the sections to dry at room temperature.

The thus-obtained mononuclear cells or cryostat sections are thensubjected to an assay that enables the determination of the number ofCD8⁺ T cells that express Vα12.1 in the sample. Detection of CD8⁺ Tlymphocytes in a population and those which are Vα12.1 positive can bedetermined simultaneously, or sequentially. The CD8⁺ subpopulation of Tcells can be isolated from other mononuclear cells by removing the CD4⁺subpopulation or alternatively, by enriching for the CD8⁺ cells. Thelatter may be accomplished, for example, by reacting cells with ananti-CD8 monoclonal antibody and capturing such cells on beads coatedwith anti-immunoglobulin. Identification of the CD8⁺ subpopulation canbe accomplished using, for example, a monoclonal antibody specific forthe CD8 cell surface antigen, such as OKT8 (Ortho Pharmaceuticals,Raritan, N.J.).

In vivo binding may be achieved by administering the antibody orfragment or derivative by any means known in the art (including but notlimited to intravenous, intraperitoneal, intranasal, and intraarterial,to name but a few) such that immunospecific binding may be detected; forexample, by attaching a radioactive label to the diagnostic antibody,fragment, or derivative.

b. Nucleic Acids

In another embodiment of the invention, an autoimmune disorder, such asRA, is diagnosed by using nucleic acid sequences that detect thepresence of Vα12.1 DNA or RNA, derived from CD8⁺ T lymphocytes.Depending on the technique to be used, either DNA or RNA probescontaining Vα12.1 sequences can be used to diagnose a subset ofrheumatoid arthritis patients. The DNA sequences for the Vα12.1 gene areknown and this information can readily be used by persons skilled in theart to design diagnostic tests to detect the Vα12.1 elevation describedherein. For sequence information, see, Sim et al., Nature, 312:771-775(December, 1984), the pertinent portions of which are herebyincorporated by reference. Samples from patients suspected of having RAcan be analyzed for the expansions of Vα12.1⁺ T cells by cDNA synthesisand polymerase chain reaction amplification, using Vα12.1-TCA-specificoligonucleotide primers. Slot blot hybridization procedure can be usedto quantitate the relative amounts of Vα12.1 containing CD8⁺ T cells.This procedure, sometimes referred to as quantitative polymerase chainreaction, is exemplified in PCT International Publication WO90/06758.

Briefly, the first step is to isolate the messenger RNA from CD8⁺ cellsfrom a patient sample. First strand cDNA is then synthesized using avirus reverse transcriptase in the presence of an oligo-(dT) primer,essentially according to published procedures (Okayama and Berg, Mol.Cell Biol., 2:161-170 (1982).

Polymerase chain reaction is then carried out in accordance withestablished techniques using primers based on published sequences (e.g.,Vα12.1 and Cα-specific primers). The PCR products are then sizefractionated, transferred, and hybridized with internal regionradio-labeled probes on a slot blot apparatus. Following hybridizationand washing, blots are dried and radioactivity counted. The results arecompared to blots obtained from CD8⁺ derived cDNA from a normal subjectthat does not have the immune disorder to determine whether Vα12.1 geneusage in expanded in the patient suspected of having RA.

An increased percentage of CD8⁺ T cells expressing Vα12.1, as evidencedby an increased intensity on the autoradiograph, is diagnostic for RA.

While the foregoing illustrates a preferred method for using nucleicacid probes as reagents in the diagnostic assay of the invention, othertechniques including Southern hybridizations will be appreciated bypersons skilled in the art and are considered within the spirit andscope of the invention.

3. Confirmatory Diagnostic Procedures

It should be understood that the diagnostic methods of the invention arebest used in the context of other diagnostic parameters in order toobtain a comprehensive patient diagnosis. A higher level of assurancecan be made in the diagnosis of RA by determining whether the expansionof Vα12.1 in a patient suspected of having the immune disorder is aclonal or oligoclonal, as opposed to a polyclonal, expansion. A clonalor oligoclonal population of T cell receptors in patients exhibiting anexpansion in Vα12.1 is indicative of an antigen-driven expansion and, inaccordance with the present invention, is further diagnostic of RA.

As used herein, a clonal expansion is one in which there is one dominantrepeated sequence including Vα12.1 and no other repeatedVα12.1-containing sequences. An oligoclonal expansion may include asmany as 2-6 different repeated Vα12.1-containing sequences, while, in apolyclonal expansion, virtually every sequence obtained is differentfrom the other sequences, with only rare repeated sequences being noted.In the case where the expansion of Vα12.1⁺ T cells is a clonal oroligoclonal one, there may also be an indeterminate number of distinctsequences present. There are a number of techniques well known topersons skilled in the art that can be used to detect a clonalexpansion, including 1) polymerase chain reaction amplification of cDNAfrom a suitable patient sample followed by sequencing and 2) Southernblot hybridization. Direct and inverse PCR reactions can be used, asdescribed in the Examples. Preferably, the nucleic acid samples used forthe clonality analyses are obtained from an enriched CD8+ population ofthe patient's peripheral blood lymphocytes.

Southern hybridization is the preferred procedure for detectingclonality of the Vα12.1 expansion when screening a large number ofpatients suspected of having RA. When only a small number of patients isinvolved, polymerase chain reaction followed by sequencing is thepreferred technique for determining whether the expansion of Vα12.1 isclonal or oligoclonal. An example of this technique for determiningclonality in a patient sample is provided in the Examples herein.

A higher level of assurance can also be made in the diagnosis of RAthrough HLA-typing. In accordance with the present invention, it hasbeen discovered that the subpopulation of RA patients exhibitingelevated Vα12.1 expression also exhibit a statistically significantincrease in the proportion of occurrence of the HLA-DQw2 allele.Association of the HLA-DQw2 genotype with an elevation in Vα12.1 bearingCD8⁺ T cells is diagnostic for a subset of patients with RA. HLA-typingis widely known and used and can be conducted in accordance with any ofthe known techniques, including serological detection and detection ofHLA-DR locus gene products by cellular techniques. See generally, Taitand Simmons, Detection of Immune-Associated Genetic Markers of HumanDisease, Chapter 4, (Churchill and Livingston, pub., 1984).

In addition, a diagnosis of rheumatoid arthritis may be made based onthe methods of the invention in the context of other clinical featuresof rheumatoid arthritis, such as typical joint involvement (chronic,symmetrical arthritis; early involvement most often in the hands);characteristic radiographic features; the presence of rheumatoid factor;the presence of rheumatoid nodules, etc. (Fishman et al., Medicine,Second Edition, J. B. Lippincott Company, Philadelphia, pp. 340-346). Aswith any diagnostic criteria, the parameters disclosed in the presentinvention may not be sole determinants, or pathognomonic, of aparticular disorder.

4. Diagnostic Kits

Diagnostic kits are useful in performing the diagnostic methodology ofthe present invention. Such kits include a Vα12.1-specific reagent,preferably an anti-Vα12.1 monoclonal antibody of the invention, or afragment or derivative thereof, coupled with an appropriate detectablemarker for the analysis, suitable standards, solid phase components suchas microscope slides, microtiter dishes, or beads, as necessary, andother components such as enzymes and substrates useful for detection.The diagnostic kits will also typically comprises a reagent capable ofidentifying the CD8⁺ subpopulation of lymphocytes, preferably ananti-CD8 monoclonal antibody, together with the appropriate reagents andlabels. The diagnostic kits of the present invention can also includeHLA-typing reagents and particularly a reagent for detecting theHLA-DQw2 allele.

5. Treatment of Rheumatoid Arthritis With Anti-Va12.1 Antibodies

In another embodiment of the invention, a Va12.1-specific reagent isadministered to a subject in order to modulate the function and numberof Vα12.1⁺ T cells in vivo. Preferably, the reagent is a monoclonalantibody, derivative, or fragment thereof specific for an epitope of theVα12.1 variable region of the TCR, although other Vα12.1 specificreagents may alternatively be employed. Although it is contemplated thatthe Vα12.1 specific reagents will have applicability for animal subjectsin addition to human beings, such as domesticated animals, thetherapeutic aspects of the invention are of the greatest value in thetreatment of rheumatoid arthritis in humans.

Modulation of Vα12.1 T cells in vivo using the Vα12.1-specificantibodies of the invention involves administering a single dose ormultiple doses of the antibody, fragment, or derivative in atherapeutically effective amount. The modulation of the Vα12.1 T cellpopulation can either be positive, resulting in an increased number andfunction of the Vα12.1 expanded population of T cells, or it can benegative, resulting in the elimination of the deleterious population ofT cells. Thus, for example, if the antibody is capable of inducing invitro T cell proliferation, and it is desirable to enhance the expandedVα12.1⁺ T cell population, the antibodies, fragments or derivatives maybe administered in an unconjugated form to increase the Vα12.1 expandedpopulation and stimulate specific cell-mediated immunity.

In most instances, however, it will be desirable to eliminate or block(i.e., negatively modulate) the Vα12.1 expanded T cell population.Therefore, in another particular embodiment, an antibody, fragment, orderivative specific for an epitope of the Vα12.1 variable region of theTCR can be used to target a cytotoxic molecule to Vα12.1 CD8⁺ cellswhich are the causative agents of the autoimmune disorder, therebynegatively effecting the function and number of these T cells. It isalso possible to use an unconjugated Vα12.1-specific antibodytherapeutically to block the interaction of effector T cells with theirspecific antigen and thus modulate a deleterious response. A furthertherapeutic embodiment is to administer an antibody therapeutically tobind to its target and mark those cells for elimination by the RESsystem or by antibody dependent cell cytotoxicity (ADCC) reactions, withthe ablation of the target T cells resulting in a therapeutic effect.

The route of administration for any of the foregoing therapeuticmodalities may include intradermal, intramuscular, intraperitoneal,intravenous, or subcutaneous injection, intranasal routes and slowrelease forms, such as those delivered in transplantable forms, onpatches or in other colloidal forms. In one embodiment, the antibody canbe encapsulated in liposomes.

When used to treat disease, the Vα12.1-specific antibodies can be usedin unmodified form for positively modulating the number and function ofthe Vα12.1 expanded T cell population or in unmodified form orconjugated to radioucleotides or toxins by means well known in the artand used to deliver the conjugated substance to deleterious T cells fornegative modulation. Non-limiting examples of radionucleotides which canbe conjugated to antibodies and administered include ²¹² Bi, ¹³¹ I, ¹⁸⁶Re, and ⁹⁰ Y. These elements exert their effect by locally irradiatingthe cells, leading to various intracellular lesions, well known topersons skilled in the art of radiotherapy.

Cytotoxic drugs that can be conjugated to antibodies and administeredfor in vivo therapy include, but are not limited to, daunorubicin,doxorubicin, methotrexate, and mytomycin C. For a more detaileddiscussion of these classes of drugs and their mechanisms of action,see, Goodman et al., Goodman and Gilman's The Pharmaceutical Basis OfTherapeutics, 8th ed. Pergamon Press (1991).

As an example of conjugation to a toxin, the 6D6 monoclonal antibody canbe combined with diphtheria toxin, by the method of Bumol, Proc. Natl.Acad. Sci., 80:529 (1983). Briefly, monoclonal antibodies reactive withthe Vα12.1 T-cell receptor segment are prepared by as described. Theantibodies are purified and combined with excess (6 mol/mol)N-succinimydyl 3-(2-pyridyldithio) propionate (Pharmacia, Uppsala,Sweden) in PBS. After 30 minutes incubation at room temperature, thesolution is dialyzed against PBS. The modified antibodies are conjugatedwith an appropriate toxin, such as diphtheria toxin A chain. Othertoxins such as ricin A can also be employed. The diphtheria toxin Achain is isolated as detailed in Bumol, supra. The modified antibodiesare mixed with excess (3 mol/mol) reduced diphtheria toxin A chain (10%of the total volume), allowed to react for 36 hours at 4° C., andconcentrated by chromatography on Sephadex G-2000. The product isapplied to a Sephadex G200 column (1.0×100 cm), allowed to equilibrateand eluted with PBS.

The effective dose of the therapeutic reagent will be a function of theparticular Vα12.1-specific reagent or fragment or derivative employed,the presence and nature of conjugated therapeutic reagent, the patient,and his or her clinical condition. Effective doses of the antibodies,fragments, or derivatives of the invention for use in preventing,suppressing, or treating an immune-related disease are in the range ofabout 1 ng to 100 mg/kg body weight. A preferred dosage range is betweenabout 10 ng and 10 mg/kg, and a more preferred dosage range is between100 ng and 1 mg/kg.

The mode of treatment of rheumatoid arthritis can involve acutetreatment conditions, and will typically also involve chronic treatmentconditions, given the nature of the disease. This in turn will lead totreatment regimens involving initial bolus administrations or continuousadministrations, followed by repeated administrations at treatmentintervals, initially approximately every three days, and laterapproximately every couple of weeks, as the severity of the diseasedecreases with the treatment.

Various pharmacologic compositions may be utilized in order to deliverthe antibodies, or fragments or derivatives thereof, according to theinvention. Any suitable pharmaceutical agent with desirable solubilitycharacteristics and chemical properties may be used, including but notlimited to, where appropriate, saline or dextrose solutions. The reagentitself must be properly formulated, for example, as a humanized orchimaeric antibody combined with various buffers, sugars, or stabilizingcompounds that increase the stability or half life of the antibody. Toextend the half-life, the reagent can first be modified to increase ordecrease the amount of carbohydrate complexed to it, or alternatively,can be complexed with a reagent such as polyethylene glycol. Finally,pharmaceutical compositions comprising the therapeutic reagent in theappropriate buffers, salts, and pH are required.

Therapeutic kits can comprise the therapeutic compositions of theinvention in one or more containers.

6. Monitoring The Effectiveness Of Therapy With Vα12.1-Specific Reagents

The same Vα12.1-specific reagents used to diagnose and/or treatrheumatoid arthritis can also be used to monitor the effectiveness of adisease therapy or to monitor the progression of the disease throughoutphases of remission, relapse, or stable periods. Thus, in rheumatoidarthritis, the initial disease correlation is made on the basis of anelevation of Vα12.1 CD8⁺ T-cells. Vα12.1-specific reagents or othertherapeutic reagents can then be used to treat the immune disorder.During the treatment course, as the deleterious T cells are beingeliminated, the Vα12.1-specific reagent can be used to monitor theextent of the elimination of the Vα12.1 positive T cells to the pointwhere the cells are gone and the disease is in remission. Following aremission, the Vα12.1-specific reagent is used periodically inrediagnosis, to determine whether the patient is stable, in remission,relapse, or is otherwise in need of treatment. The Vα12.1-specificreagents, especially the monoclonal antibodies of the invention, arethus useful in combined diagnostic and therapeutic procedures.

Deposit Information

Samples of the hybridoma cell line (designated herein as 6D6) thatsecretes anti-Vα12.1 murine monoclonal antibody were deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,on Aug. 21, 1991 under the terms of the Budapest Treaty and assignedATCC accession number HB 10858. Without admitting that access to thehybrid cell line is necessary to practice the claimed invention, it isagreed that, upon allowance and issuance of a patent for this invention,all restrictions on the availability of the culture deposit designatedherein will be removed and the designated culture will be maintainedthroughout the effective life of the patent granted, for 30 years fromthe date of deposit or for five years after the last request for thedeposit after issuance of the patent, which ever is longer.

The invention will be more fully understood from the following Examples.

EXAMPLE 1

This example describes the preparation of the anti-Vα12.1 monoclonalantibody designated 6D6.

Establishment of Hybridomas

T cell receptor protein from the HPB-MLT human leukemia cell line wasisolated by immunoprecipitation from Triton X-100 (TX-100) lysates ofHPB-MLT. For each immunization, 0.5 g of lymphocytes was solubilized in10 ml of 1% TX-100, was immunoprecipitated with 3 μl of T40/25 ascites,and was adsorbed to 125 μl of 10% (w/v) Staphylococcus aureus Cowan Istrain (SACI) (Calbiochem, La Jolla, Calif.). The adsorbed immunecomplexes were injected i.p. in phosphate-buffered saline (PBS), pH 7.2into 8-wk-old BALB/c mice at monthly intervals. Mouse sera obtained bytail bleeding were tested by immunoprecipitation on ¹²⁵ I-labeledHPB-MLT cell lysates to detect the presence of clonotypic or V geneencoded specific mAb and on ¹²⁵ I-labeled human PBL lysates to detectthe presence of mAb against TCR unrelated to HPB-MLT.

Early in the course of immunization, clonotypic monoclonal antibodiesand V gene encoded specific monoclonal antibodies could be detected.Immunizations were conducted every two to three weeks for a total periodof about six months. To diminish the proportion of mAb directed againstthe SACI immunoabsorbent, a final i.v. injection of immunoaffinitycolumn-purified HPB-MLT TCR was performed. Briefly, a crude membranepreparation was made from frozen HPB-MLT cells after solubilization in1% Nonidet P-40. Glycoproteins from this extract were purified by lectinaffinity chromatography and passed over a column of mAb T40/25 coupledto Sepharose CL-4B (Pharmacia, Pescataway, N.J.). The bound proteinswere eluted from the column with 50 mM diethylamine, pH 11.5 in 0.1%deoxycholate, and the eluted material was neutralized, dialyzed againstPBS to remove this detergent, and 0.1% TX-100 was added before injectionof 1 μg of the purified HPB-MLT TCR protein. Three days after the i.v.boost, the mouse was sacrificed and spleen cells were removed. Immunespleen cells (2×10⁸) were fused with myeloma P3X63Ag8.653 cells in thepresence of polyethylene glycol 1500 (British Drug House, Carlplace,N.Y.). The fused cells were then selected in the presence ofhypoxanthine/aminopterin/thymidine (Sigma, St. Louis, Mo.) in RPMI 1640(GIBCO, Grand Island, N.Y.) containing 15% fetal calf serum in 24-welltissue culture plates (Flow, McLean, Va.).

Supernatants from wells positive for growth were screened byimmunoprecipitation on ¹²⁵ I-labeled HPB-MLT cell lysates by adding mAb187.1 (rat anti-mouse_(K)) to insure protein A binding of the mousehybridoma antibodies. Twenty-three wells produced supernatants thatimmuno-precipitated the TCR complex from HPB-MLT cells. These were thenexamined for their ability to immunoprecipitate from and stain HPB-MLT,PBL and other cell lines bearing the same TCR V genes expressed onHPB-MLT cells.

One of the hybridomas that specifically immunoprecipitated HPB-MLTexhibited reactivity with about 2% to 5% of peripheral T cells fromnormal individuals, as determined by cytofluorographic analysis. SeeFIG. 1 herein. This monoclonal antibody was given the designation 6D6,and subcloned for further study in accordance with conventionaltechniques.

Reactivity Of 6D6 mAb 6D6 Reacts Specifically With The T Cell ReceptorOf HPB-MLT

To determine the nature of the molecule recognized by mAb 6D6,immunoprecipitations were performed on ¹²⁵ I-labeled HPB-MLT celllysates followed by SDS-PAGE and autoradiography by using Na ¹²⁵ I andlactoperoxidase as previously described. Brenner, Nature, 322:145(1986). As illustrated in FIG. 2, the mAb immunoprecipitated the TCR αβheterodimer (85 kd) under non-reducing conditions (FIG. 2A, lane 4). The85 kd heterodimer resolved into 2 species with Mr 46 kD (TCRα) and 39 kD(TCRβ) under reducing conditions (FIG. 2A, lane 8). For comparison,similar radiolabeled species were visualized after immunoprecipitationswith TCR β-specific mAb βF1 and CD3ε-specific mAb Leu 4 (FIG. 2A). Inboth anti-TCR and anti-CD3 immunoprecipitations, a variable degree ofco-immunoprecipitation of the other components was noted. Together,these data indicate that the 6D6 monoclonal antibody is specificallyreactive with theft cell receptor of the HPB-MLT clone.

6D6 Is Specific For A Determinant Expressed On The TCR α Chain

To delineate the TCR chain specificity of mAb 6D6, immunoprecipitationswere carried out on HPB-MLT cell lysates metabolically labeled with ³⁵[S] methionine and cysteine.

Biosynthetic labeling of TCR chains from HPB-MLT was carried out asfollows. Cells were washed and incubated at 5×10⁶ cells/ml inmethionine- and cysteine-free RPMI-1640 containing 10% dialyzed fetalcalf serum (FCS), 2 mM glutamine and 20 mM HEPES at 37° C. in 5% CO₂atmosphere. After 30 minutes, 0.5 mCi each of [³⁵ S]-methionine and-cysteine were added and incubation continued for 4 hrs. Cells werepelleted and lysed in Tris-buffered saline (TBS, 50 mM Tris pH 7.5, 150mM NaCl) containing 1% Triton X-100, 1 mM PMSF and 8 mM iodoacetamide.Cell lysates were precleared twice using normal rabbit serum (NRS) andfixed Staphylococcus aureus Cowan I (Pansorbin, Calbiochem-BoehringCorp., San Diego, Calif.). Specific immunoprecipitations were carriedout using amounts of ascites determined to be optimal; 6D6 (0.1 μl), 3A2(anti-vβ 5.3)(0.4 μl), Leu 4 (anti-CD3)(0.1 μl), βF1 (anti TCR Bframework)(0.25 μl) or αF1 (anti-TCR a framework)(0.1 μl). In each case150 μl of mAb 187.1 (rat anti-mouse K) culture supernatant was added asa second antibody. After 60 min incubation at 4° C. 100 μl of 10%Protein-A sepharose CL4B (Pharmacia Fine Chemicals, Sweden) was addedand incubated further for 30 minutes. Immune complexes were washed in0.1% Triton X-100, resolved by SDS-PAGE and analyzed by autoradiographyas described (Laemmli, Nature, 227:680 (1970)).

Under these conditions, a detectable fraction of the newly synthesized αand β TCR chains were still unpaired, such that mAb 6D6immunoprecipitated both a free α chain (migrating as a sharp band at 43kD) and the αβ-heterodimers as fully glycosylated (80-85 kD) andpartially glycosylated (70-75 kD) structures (FIG. 2B, lane 5) based onsizes reported earlier (Alarcon et al, J. Biol. Chem., 263:2953 (1988)).Immunoprecipitation with the anti-TCR α framework antibody, αF1,confirmed that the 43 kD species was the unpaired TCR α chain subunit(FIG. 2B, lane 4). The other radiolabeled species (41-44 kD) in the αF1precipitation presumably corresponded to differentially glycosylatedα-chain species, as reported previously. By comparison, other mAbs thatwere generated including the anti-TCR β framework antibody, βF1 (lane2), and antibody against the Vβ product of HPB-MLT (3A2) (lane 3) bothimmunoprecipitated the unpaired β-chain ( 38 kD) but not any of the freeα-chain species. It was thus determined that mAb 6D6 reacted with adeterminant expressed on the TCR-α chain.

The Determinant Recognized By 6D6 mAb Is Encoded By The Vα12.1 GeneSegment

To examine whether the determinant recognized by 6D6 was encoded by Vα,Jα, or a combination of both gene segments, 6D6⁺ clones from healthyindividuals were analyzed. Peripheral blood mononuclear cells wereseparated from heparinized blood from healthy blood donors by FicollHypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) gradientcentrifugation, washed twice in RPMI 1640 medium (Gibco, Paisley, UK),macrophage depleted and diluted in phosphate buffered saline. 6D6⁺clones were then subcloned as follows.

Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with100 μg/ml-of immobilized mAb 6D6. These cultures were supplemented withconditioned media containing I1-2 after five days of stimulation withthe 6D6 mAb. Cells were restimulated with 6D6 mAb (100 μ/ml) andautologous PMBC (4000 rads) two to three weeks after initialstimulation. Within a four or five week period, these cultures containedup to 80% 6D6 positive T cells versus the 3 to 4% in the unstimulatedPBL. Cloning and subcloning was achieved by limiting dilutions in mediacontaining IL-2 and a panel of 6D6⁺ and 6D6⁻ T-cell clones was derivedby staining with 6D6 mAb and other T cell markers. 6D6⁺ T cells clonesincluded HD5.A and HD5.B.

Genomic DNA was then extracted from the 6D6⁺ T cell clones HD5.A andHD5.B and a 6D6⁻ clone, 3A2.D using a standard technique. (Wigler et al,Cell, 16:777 (1979)). Briefly, lymphocytes were ficolled to remove deadcells. After PBS wash, cells were transferred to 15 ml Sarstedt tubesand resuspended into 100 μl PBS. Cells were then solubilized in lysisbuffer (1X TNE, 0.4% SDS and 100 μg/ml Proteinase K, 2.5 mls for 10⁷cells) by vigorous pipetting. Tubes were rotated overnight at 37° C. Thesolubilized cells were then extracted once with 1 volume of chloroform.Two volumes of absolute ethanol were added to precipitate the DNA.

Precipitated DNA was then transferred by plastic pipette to cold (-20°C.) 70% ethanol and washed. After drying the DNA, 900 μl of H₂ O andtubes rocked for two hours, after which 100 μl of 10X TE buffer wasadded. The optical density at 260 was measured to calculate DNAconcentration.

The thus-prepared DNA was digested with the restriction enzyme BamHI. 10μg of DNA was digested overnight in 400 μl of buffer using 50 units ofthe restriction enzyme. To complete digestion, an additional 50 unitunits of enzyme was added and the digestion continued for another threehours. Two volumes of absolute ethanol were added at -20° C. for twohours to precipitate the DNA, making sure salt content was sufficientfor precipitation. DNA was then spun and washed once with 70% ethanoland dried in a speed vacuum.

The digested DNA was then resuspended in 36 μl TE buffer, heated forfive minutes at 56° C. and 4 μl 10X sample buffer added for loading ontoagarose gel for Southern transfers.

5 μg DNA were loaded into a lane of a 0.7% agarose gel andelectrophoresed in 1 x TBE buffer. Electrophoresis was carried out at 35volts for 15 hours. The gel was then photographed and denatured for onehour at room temperature in 1.5M NaCl containing 0.5N NaOH. The gel wasthen reneutralized by washing three times in 1.5M NACl with 1M Tris, pH8. DNA was then transferred from the agarose gel to Hybond-N nylonmembrane (AMERSHAM) overnight in 20 X SSC. The membrane was then rinsedin 6X SSC and DNA was crosslinked to the membrane by UV irradiation (4min).

Vα12.1 DNA was radiolabeled by hexapriming using 100 μg of DNA insert,(pGA5, Sim et al, previously cited). The Vα12.1 probe was an ECO-RI--ACCI fragment of the TCR α chain cDNA clone pGA5. Hybridization was carriedout using standard conditions. Briefly, approximately 10⁶ cpm/lane wasincubated in sealed plastic bags containing 50% formamide, 6% SSC, 5 xDenhardt's reagent, 0.5% SDS, 50 Mm Hepes, and 200 μg of ssDNA (salmonsperm) overnight at 42° C. Blots were washed once in 1x SSC containing0.5% SDS at 42° C. (or 50° C). Autoradiography was carried out byexposing the blots to x-ray film at -70° C.

As shown in FIG. 3, the germ line configuration (GL) for the Vα12.1 genewas seen as a 6.3 kb fragment in the β cell, SD (lane 1). Both Vα12.1alleles in 3A2 D T cell clone were deleted, while DNA from the 6D6⁺clones HD5.A and HD5.B and that from the HBP-MLT line showed distinctrearrangements.

The fact that the rearranged fragments were of different sizes in eachcell line indicated that the Vα12.1 gene segment, which is part of a Vfamily having only one member, might be rearranging to several differentJα segments. This suggested that mAb 6D6 was recognizing a Vα ratherthan a Jα gene segment encoded determinant.

In order to understand the relationship between Vα12.1 gene usage andthe expression of the determinant recognized by 6D6, alpha chainjunctional sequences from three 6D6⁺ T cell clones (HD5.A and HD5.B andHPB-MLT) were sequenced.

To clone and sequence the junctional sequences of the 6D6+ T cell clonealpha chains, Vα12.1 and Cα specific oligonucleotide primers were usedin polymerase chain reaction (PcR). These products were cloned into M13phage DNA by using cloning sites synthetically introduced into the PCRprimers.

The ligated M13 phage DNA was cloned by plating into JM101 bacterialcell. Phage plaques containing PCR products were isolated and LB brothcontaining 1/100 dilutions of JM101 cells were inoculated. Phage weregrown for 4.5 hours at 37° C. 1.5 mls were then poured into and spun inmicrofuge tubes and spun for five minutes at room temperature. 1.2-1.3ml of supernatants containing 150-200 μl 20% polyethylene glycol (PEG)containing 2M NACl and magnesium sulfate (MS) were pipetted and cellsmixed and incubated at room temperature for 15 minutes. The phagepellets were spun down and the supernatant discarded. Pellets were thenresuspended in 100 μl of 10 mN Tris pH 8.0 with 0.1 mM EDTA andextracted with 100 μl of phenol equilibrated with 10 mM Tris 8.0, 0.1mMEDTA. Supernatant was added to a tube containing 300 μl of ethanol with3M sodium acetate. Incubation was conducted for two hours at 20° C.,followed by spinning for 15 minutes at 4° C. The DNA pellets were thenwashed and dried in a speed vacuum and resuspended with 30 μl of 10 mMTris 8.0 and 0.1 mM EDTA. Phage yield was 5-10 μg per mini prep.

Sequencing was conducted by the dideoxy chain termination method usingmodified T7 polymerase (Sequenase, United States Biochemical Corp,Cleveland, Ohio). 5 μl of phage DNA containing inserts were used in thesequencing by annealing a universal primer to the M13 sequences close tothe insertion site and the appropriate amount of deoxynucleotidesprovided in combination with one of four deoxynucleotide triphosphateanalogs that terminate DNA extension. Each of the four separatereactions were analyzed in separate lanes on sequencing gels. Thepatterns of dideoxynucleotide incorporation were visualized byautoradiography due to the incorporated deoxynucleotide. Sequences wereanalyzed and Vα12.1 and Cα specific sequences were lined up withpublished sequences.

The results of the sequencing analysis revealed that each of the three6D6⁺ T cell lines contained in frame Vα12.1 rearrangements to distinctJα gene segments. These results demonstrate that the mAb 6D6specifically recognized the product encoded by the Vα12.1 gene segment.

EXAMPLE 2

This example determines the percentage of expression of the Vα12.1 genesegment in peripheral blood T-cell subsets in normal, healthyindividuals.

Peripheral blood samples were obtained from 20 healthy adult blooddonors. Peripheral blood mononuclear cells (PBMC) were then separatedfrom heparinized peripheral blood by Ficoll-Hypaque (Pharmacia FineChemicals, Uppsala, Sweden) gradient centrifugation. Heparinized bloodwas also obtained from the umbilical cords of 10 newborn humans ofuncomplicated deliveries at the Brigham and Women's Hospital.

The PBMC and CBMC from each of the subjects were analyzed by a two colorstaining procedure, to determine the percentage of 6D6⁺ T cells in CD4⁺and CD8⁺ subsets.

All cells were washed in tissue culture media (RPMI-1640) containing 10%fetal calf serum. (FCS). Isolated PMBC or CBMC were suspended inphosphate buffered saline containing 2% pooled human serum and 0.2%sodium azide (staining buffer). Fifty μl of cell suspension containing10⁵ PMBC or CBMC were incubated with directly conjugated antibodies byadding an equal volume of antibody premix containing 5 μg/ml ofbiotinylated 6D6 (red fluorescence) with 5 μg/ml of fluoresceinisothiocyante (FITC)-OKT4 (anti-CD4, Ortho Pharmaceuticals, Raritan,N.J.) or FITC-OKT8 (anti-CD8, Ortho) monoclonal antibodies (greenfluorescence). After one hour of incubation on ice, cells were washedtwice and resuspended in 50 μl of Streptavidin-Phycoerytherine (PE) andincubated for 30 minutes to allow biotin-avidin binding. After 30minutes the cells were washed thrice and resuspended in 400 μl ofstaining buffer containing 1.25 ng/ml of propidium iodide (PI). Twocolor FACS analysis was then carried out with a FACSCAN (BectonDickinson, Mountain View, Calif.).

Lymphocytes were gated on the basis of forward and side scatter and deadcells were gated out by staining with propidium iodide and 10⁴ cellswere analyzed for fluorescence. Optimal compensation was set for greenand orange fluorescence.

The results of an analysis of a peripheral blood sample from a normal,healthy blood donor for Vα12.1 expression among CD4⁺ and CD8⁺ T cells bytwo color staining is shown in FIG. 4A. In this Figure, PBMC from anadult were stained with FITC-OKT4 and FITC-OKT8 (green fluorescence) andanti-Vα12.1 (6D6) (red fluorescence) and analyzed as described above.

Dot plots were divided into quadrants to represent unstained cells(lower left, quadrant 3), cells stained with FITC alone (lower right,quadrant 4), cells stained with PE alone (upper left, quadrant 1) andcells that double stained with FITC and PE (upper right, quadrant 2).

The Vα12.1 expression in CD4⁺ and CD8⁺ T lymphocytes from adult andnewborn cells (see FIG. 4B) was determined using data for individualblood samples stained as in FIG. 4A. The expression of Vα12.1 in theCD4⁺ and CD8⁺ subsets for each individual are connected by a line. Thefollowing formula was used in the calculation of percentage values for6D6 reactivity with CD4⁺ or CD8⁺ T cells. ##EQU1##

As illustrated in FIG. 4A and 4B, when Vα12.1 gene expression wasanalyzed among the CD4⁺ and CD8⁺ T cells by two color staining, Vα12.1was expressed on about 1.4 to about 3.75% (mean 2.66%±0.68%) of CD4⁺ Tcells of normal healthy adult blood donors, while Vα12.1 was expressedon about 1.4 to about 7.2% (mean 4.2±1.62%) on the CD8⁺ T cells of thesame individuals. Most individuals therefore expressed a 2-3 fold higherfrequency of Vα12.1 among CD8⁺ compared to CD4⁺ T cells. This was alsotrue in newborns. See FIG. 4B. Vα12.1 was expressed on about 1.7 toabout 4.1% (mean 2.94%±0.74%) of CD4⁺ T cells of normal newborns, whileVα12.1 was expressed on about 3.0 to about 8.8% (mean 5.92±2.03%) on theCD8⁺ T cells of the same newborns.

EXAMPLE 3

This example demonstrates an expansion of Vα12.1 gene usage in a subsetof rheumatoid arthritis patients.

Expression of Vα12.1 gene usage in a total of 80 patients with severalautoimmune diseases, including rheumatoid arthritis (46 patients),osteoarthritis, polymyosistis, and systemic lupus erythematosus, wasexamined using the Vα12.1 mAb as in Example 2, except that peripheralblood samples were obtained from patients showing clinical symptoms ofthe respective diseases. Briefly, flow cytometric analyses wereperformed using directly conjugated antibodies. Heparinized blood wasobtained from individuals and PBMC were isolated using Ficoll-Paque(Pharmacia Fine Chemicals, Uppsala, Sweden) and suspended in stainingbuffer (PBS/2% human serum with 0.02% NaN₃) containing saturatingamounts of conjugated mAbs and analyzed by a Facscan Flow cytometer(Becton Dickinson). Two-color immunofluorescence analysis of Vα12expression on CD4⁺ and CD8⁺ T cells was carried out on peripheral bloodmononuclear cells (PBMC) using OKT4 (anti-CD4) or OKT8 (anti-CD8) FITCconjugated mAb and anti-Vα12 mAb (6D6-PE) as previously described usinga FACSCAN (Becton Dickinson and Co., Mountainview, Calif.). Lymphocyteswere gated on the basis of forward and side scatter profiles andanalyzed for fluorescence intensity in log scale. Ten thousand viablecells were analyzed by gating on lymphocytes excluding propidium iodide.The data were analyzed by dividing the dot plot into quadrants torepresent unstained cells, cells stained with FITC alone (OKT4 or OKT8),cells stained with PE alone (6D6), and cells that were co-stained withFITC and PE. The Vα12 expression on CD4⁺ and CD8⁺ T cells from adult RApatients was determined using data generated for individual peripheralblood samples stained as in Example 2. The following formula was used tocalculate the percentage value for Vα12 expression: % Vα12⁺ /CD4⁺ (orCD8⁺) cells={Vα12⁺ cells co-stained with anti-CD4 (or anti-CD8) (2ndquadrant)/% T cells that were CD4⁺ (or CD8⁺)}×100. The results aresummarized in FIG. 5.

As illustrated in FIG. 5, one group of patients contained percentages ofVα12.1 bearing CD8⁺ T cells (mean value of 3.6%, range 1.0% to 7.0%)which was similar to those found in normal subjects. However, thedistribution was bimodal, as a second group of patients had much higherpercentages of Vα12.1⁺ T cells (mean value of 22%, range 8.0% to 43%) inthe CD8⁺ subset. This Vα12-elevated group of patients had greater thanthe 2×SD (7.3%) of the mean in healthy individuals tested (3.6%). Eachof the eight patients in this group, sometimes hereinafter referred toas the Vα12-elevated group, contained 43%, 29%, 22%, 27%, 26%, 20%, 12,and 9% Vα12.1 bearing CD8⁺ T cells, respectively. The high percentagesof Vα12.1⁺ cells in these patients was a relatively stable phenomenon aspatients tested over approximately a one year period showed consistentlevels. The increased numbers of Vα12.1 cells were not noted in the CD4⁺subpopulation of T cells.

EXAMPLE 4

To gain insight into the basis for the Vα12.1 T cell expansion in RA,Vα12.1 transcripts from selected CD8⁺ T cells from three patientsexhibiting the expansion were cloned and sequenced. Nucleic acidsequences of Vα12.1-containing transcripts were generated by direct(Vα12.1-specific) or inverse polymerase chain reaction (PCR) (all Vα's).

Isolation of CD8+, Vα12.1+ Cells

CD8⁺, Vα12.1⁺ T lymphocytes from two RA patients exhibiting an expansionof Vα12.1 were obtained from the patient's peripheral blood and isolatedusing a two-step selection procedure. Separation of the CD4⁺ and CD8+ Tcells was carried out by Ficoll-Paque (Pharmacia Fine Chemicals,Uppsala, Sweden) on the isolated cells, which were suspended inRPMI-1640 containing 5% fetal calf serum (FCS) at 5×10⁵ /ml for mixingwith saturating amounts of anti-CD4 (OKT4) monoclonal antibodies (mAb).The first selection was carried out by rotating the cell suspensions for1 hour at 4° C. and washing away free antibody with RPMI/5% FCS beforeadding a 1 to 2 fold excess of goat anti-mouse Ig conjugated Dynabeads(Dynabeads M-450, Dynal, Oslo, Norway). Cells bound to the beads (CD4⁺)were removed. CD4-depleted populations of cells were reselected twicemore with beads to remove completely any residual CD4⁺ T cells. Underthese conditions, the CD8⁺ T cells were enriched to greater than 98%purity, as verified routinely by staining the negatively selectedpopulations by FACS. A second step of selection with anti-Vα12.1 (mAb6D6) was carried out on the CD8-enriched cells in order to isolateVα12.1⁺ /CD8⁺ T cells. Double selected cells (Vα12.1⁺, CD8⁺) are washedextensively before solubilizing in RNA lyses buffer for RNA isolationfor use in the polymerase chain reactions.

RNA Purification

Purification of RNA from CD8⁺ or Vα12.1⁺ /CD8⁺ T cells was carried outaccording to Chomczynski and Sacchi, Anal. Biochem., 162:156 (1987).Total RNA was quantitated and analyzed for intactness by resolution on a1% agarose minigel and visualization upon staining with ethediumbromide.

Direct Polymerase Chain Reaction

Complementary DNA (cDNA) for the direct PCR method was synthesized at42° C. in 50 μl reactions using 500 ng of oligo-(dT)₁₂₋₁₈ primer, 1 to 5μg total RNA and 10 U AMV reverse Transcriptase (Promega Corp., Madison,Wis.). After 1 hour, the reaction mixture was diluted to 100 μl, boiled,chilled, and centrifuged to remove insoluble material. PCR was performedin 25 μl reactions containing 1 μl cDNA (1/100), 1 mmMgCl₂, 10 mM TrispH 8.3, 1 mg.ml gelatin, 5 picomole of each primer, 0.2 mM of each dNTPand 2 U of Taq Polymerase (Thermus aquaticus DNA polymers,Perkin-Elmer-Cetus Corp.).

Briefly, cDNA and primers were pre-mixed with MgCl₂ /Tris/gelatin bufferand heated at 95° C. for 7 min. Other components were added, thereaction mixture was overlain with mineral oil, and 25 to 35 cycles ofPCR were carried out in a thermocycler (Perkin-Elmer-Cetus Corp.) at thefollowing settings: 0.7 min. at 95° C. for denaturation, 1 min. at 56°C. for annealing, and 1 min. at 72° C. for chain extension. To ensurecomplete synthesis, the last cycle at 72° C. was extended to 10 min. TheVα12.1 and Cα primers used were the same as those used and illustratedin DerSimonian et al., J. Exp. Med., 174:639, 640 (September, 1991).See, also Sim et al., Nature, 312:771-75 (December 1984) for thecomplete nucleic acid sequence of the TCR α chain from HPB MLT. The SalI and HindIII restriction sites in the respective PCR primers were usedto generate sticky ends for cloning.

Inverse Polymerase Chain Reaction

Inverse PCR (iPCR) was first established to amplify the flanking regionof a known sequence within a given restriction fragment of genomic DNA.Recently, this method was shown to be applicable to the amplification ofthe full length cDNA of TCR α and β genes. Uematsu, Immuno-genetics,34:174 (1991); Uematsu et al., Proc. Natl. Acad. Sci. U.S.A., 88:8534(1991).

To identify all of the Vα and Jα gene segments present in the Vα12.1⁺/CD8⁺ T cells, we used the iPCR method, which does not require priorsequence information on both ends of the DNA to be amplified. Thistechnique independently confirmed our findings generated by the directPCR method.

First strand cDNA for iPCR was synthesized using RNA from CD8⁺, Vα12.1⁺T lymphocytes by priming with an oligo-(dT)₁₂₋₁₈ mer and utilizingmoloney murine leukemia virus-derived reverse transcriptase(MMLV-RT)lacking RNase H activity (Gibco BRL). For oligo-dT priming, 1μg total RNA (1 μg/μl) was mixed with 0.5 μl oligo-(dT)(100 ng/μl) in10.5 μl water, incubated at 65° C. for 10 minutes, rapidly chilled at 4°C. and then spun down. For first strand synthesis, 4 μl of 5X reactionbuffer, 2 μl of 0.1M DDT and 1 μl of 10 mM dNTP were added to the primedRNA mixture, the thus-prepared mixture was then vortexed gently andpreincubated at 45° C. for 2 minutes. 1 μl (200 units) of MMLV-RT (RNaseH⁻) was added and the mixture incubated at 45° C. for 60 minutes.

For second strand cDNA synthesis, RNase H was used to nick the mRNAstrand of the RNA/DNA hybrid, generating a series of RNA primers thatserved to synthesize the second strand DNA with E. coli DNA polymerase Iand E. coli DNA ligase. The double-stranded cDNA thus prepared was bluntended using the 3'-5' exonuclease activity of T4 DNA polymerase of DNAwas circularized using T4 DNA ligase. This circular cDNA was used as thetemplate for the iPCR amplification by using a pair of Cα primers, whichwere oriented in an outward direction from one another.

Briefly, 1 μl of the circularized DNA was admixed with 34.5 μl water, 5μl 10X PCR buffer; 5 μl of 20 mM MgCl₂ ; 1 μl of dNTP (10 mM each) 1.5μl of each primer (10 pM/μl) and 0.5 μl Taq polymerase (5 u/μl) for atotal of 50 μl and the mixture was overlain with mineral oil. Thirtyfive cycles of PCR were carried out in a thermocycler(Perkin-Elmer-Cetus Corp.) at the following settings: 0.5 min. at 95° C.for denaturation, 0.5 min. at 62° C. for annealing, and 1 min. at 72° C.for chain extension. To ensure complete synthesis, the last cycle at 72°C. was extended to 10 min.

After PCR, 5 μl of 100 mMMgCl₂ and 0.5 μl of E. coli DNA polymerase I(Klenow fragment) were added to the mixture and incubated for 30 min. at37° C. PCR products were then checked on a minigel in accordance withestablished protocols.

This method generated PCR products of approximately 700 base pairslength for the TCRα transcripts. This method generated up to 2×10⁶primer specific cDNA clones starting with as little as 1 μg of total RNA(Uematsu 1991). In our experience, we were able to generate a library ofTCR α cDNA products from RNA isolated from as little as 2×10⁴ of freshperipheral T cells. The Cα forward primer contained an artificial Sal Isite and the Cα inverse primer contained a Not I site, which facilitatedDNA cloning and sequencing.

The buffers used in the inverse PCR reaction are as follows:

5X 2nd-Strand Buffer

94 mM Tris-HC1, pH 6.9, 453 mM KCl, 23 mM MgCl₂, 750 μmβ-Nicotinamidedinucleotide, 50 mM ammonium sulfate;

5X Circularization Buffer

250 mM Tris-HC1, pH 7.6, 50 mM MgCl₂, 25% w/v polyethylene glycol (PEG)8000, 5 mM ATP, 5 mM DTT;

10X PCR Buffer

670 mM Tris-HC1, pH8.8, 166 mM (NH₄)₂ SO₄, 1 mg/ml BSA.

Cloning and Sequencing of PCR Products

Appropriately sized DNA products were isolated from low melting point(LMP) agarose gels (BRL, Gaithersburg, Md.) and ligated to the M13vector with cloning sites. Cloning and sequencing were carried out bythe dideoxy chain termination method using the modified T7 polymerase(Sequenase; United States Biochemical Corp., Cleveland, Ohio) Sanger etal., Proc. Natl. Acad. Sci. USA, 74:5463 (1977). The sequencing productswere resolved on polyacrylamide gels and autoradiography was carried outaccording to standard methods.

Clonality of the Vα12.1 Expansion

The cDNA sequences of the Vα12.1 containing-transcripts are set forth inTable 1 below and also in the Sequence Listing. Sequences comprise themost 3' nucleotides (11 to 14) of the variable region Vα12.1 andcomplete Jα gene segments. All sequences are presented in the 5' to 3'direction. The 3' Vα12.1 and Jα-containing sequences are separated forin Table 1 for the sake of illustration.

                                      TABLE 1                                     __________________________________________________________________________    DIRECT PCR:                                                                   Patient 1-                                                                    3' Vα12.1:                                                                       TGTGCTCTGAGTGA-                                                      Jα Sequence:                                                            CGGCTATGGTCAGAATTTTGTCTTTGGTCCC                                                        ZGGAACCAGATTGTCCGTGCTGCCCTAT                                         # of Clones:                                                                           15/15                                                                Vα/Jα Usage:                                                               Vα12.1/JαA1                                              Patient 2-                                                                    3'Vα12.1:                                                                        TGTGCTCTGAGTGA-                                                      Jα sequence:                                                            TTATCAGGGCGGATCTGAAAAGCTGGTCTTT                                                        GGAAAGGGAATGAAACTGACAGTAAACCCATAT                                    # of Clones:                                                                           9/16                                                                 Vα/Jα Usage:                                                               Vα12.1/JαA12                                             Patient 3-                                                                    3' Vα12.1:                                                                       TGTGCTCTGAG-                                                         Jα Sequence:                                                            AGGGGGAGGTGCTGACGGACTCACCTTTGGC                                                        AAAGGGACTCATCTAATCATCCAGCCCTAT                                       # of Clones:                                                                           16/24                                                                Vα/Jα Usage:                                                               Vα12.1/JαA6                                              3' Vα12.1:                                                                       TGTGCTCTGAGTGA-                                                      Jα Sequence:                                                            GCCTTATTCAGGAGGAGGTGCTGACGGACTC                                                        ACCTTTGGCAAAGGGACTCATCTAATCATCC                                               AGCCCTAT                                                             # of Clones:                                                                           3/24                                                                 Vα/Jα Usage:                                                               Vα12.1/JαA6                                              INVERSE PCR                                                                   Patient 1-                                                                    3' Vα12.1:                                                                       TGTGCTCTGAGTGA-                                                      Jα Sequence:                                                            CGGCTATGGTCAGAATTTTGTCTTTGGTCC                                                         CGGAACCAGATTGTCCGTGCTGCCCTAT                                         # of Clones:                                                                           9/9                                                                  Vα/Jα Usage:                                                               Vα12.1/JαA1                                              Patient 3-                                                                    3' Vα12.1:                                                                       TGTGCTCTGAG-                                                         Jα Sequence:                                                            AGGGGGAGGTGCTGACGGACTCACCTTTGG                                                         CAAAGGGACTCATCTAATCATCCAGCCCTAT                                      # of Clones:                                                                           21/27                                                                Vα/Jα Usage:                                                               Vα12.1/JαA6                                              3' Vα12.1:                                                                       TGTGCTCTGAGTGA-                                                      Jα Sequence:                                                            GCCTTATTCAGGAGGAGGTGCTGACGGACT                                                         CACCTTTGGCAAAGGGACTCATCTAATCAT                                                CCAGCCCTAT                                                           # of Clones:                                                                           5/27                                                                 Vα/Jα Usage:                                                               Vα12.1/JαA6                                              __________________________________________________________________________

As illustrated in Table 1 of this Example, in each of the three patientsanalyzed, we identified repeated Vα12.1-containing clones correspondingto distinct functional TCR α chain transcripts. For example, in patient#1 where 43% of the CD8⁺ T cells were Vα12.1⁺, all 15 DNA clonesanalyzed by direct PCR had identical sequences. Similarly, 9 of 16 (56%)Vα12.1 containing DNA clones were identical in patient #2, where 26% ofthe PBL CD8⁺ T cells were Vα12.1⁺. In patient #3 where 28% of theperipheral CD8⁺ T cells were Vα12.1⁺, we identified 2 repeatedsequences, one was represented in 16 of 22 DNA clones (73%), and asecond repeated sequence was identified in 4 of 22 DNA clones (18%). Theremaining two clones were identified only once. Although the junctionalsequences were different, both of these Vα12.1 encoded sequences in thispatient used the JαA6 gene segment. Furthermore, the independent use ofinverse PCR (iPCR) to clone and sequence Vα-containing transcripts inCD4 depleted and positively selected for Vα12.1 bearing T cellpopulations was also assessed. This permitted the amplification of allTCR Vα transcripts present in Vα12.1⁺ /CD8⁺ selected T cells. By thismethod, 9 of 9 DNA clones (100%) from patient #1 were identical, butdistinct from a repeated sequence found in 21 of 27 DNA clones (78%)from patient #3. These sequences match exactly the repeated sequencegenerated by the direct PCR method, respectively. Moreover, the secondclonal population of Vα12.1 to JαA6 recombination as seen in patient #3(by the direct method) was also independently confirmed by the iPCRmethod, where 5 of 27 DNA clones (19%) were found to be identical. Thus,two independent methods of generating TCR Vα specific PCR productsvalidate each approach and confirm the oligoclonality of Vα12.1expansion in RA.

Notably, all of the repeated Vα12.1⁺ T cell rearrangements in the 3patients analyzed use either JαA1 (patient #1), JαA12 (patient #2) orJαA6 (patient #3) each of which encodes a common sequence at the 3' endof Jα gene segment. This short stretch of shared residues (pro-tyr) ispredicted to contribute (or is immediately adjacent) to the thirdcomplementarily determining region (CDr3) and thus may play a role inantigen or MHC recognition. Interestingly, only six of the 80 known Jαgene segments encode this two amino acid sequence stretch. The strikingoccurrence of repeated sequences found in these patients was notobserved in similar analysis of normal subjects, and it suggests acorresponding clonality of Vα12.1⁺ T cells in the patient's peripheralblood.

EXAMPLE 5

This Example examines the surface expression of several activationmarkers on the peripheral Vα12.1 T cells in RA patients and demonstratesthat the expanded Vα12⁺ T cells in RA were previously activated.

The cell surface expression of DR, IL-2R α and β chains, CD45RO, VLA-1,and transferrin receptor on CD8+,Vα12.1⁺ cells from three RA patientswere analyzed as described in Example 3. Ma/Abs LB3.1 (anti-HLA DR),B1.49.9 (anti-IL-2Rα), Tu27 (anti-IL-2RB), UCHL1 (anti-CD45RO), TS2/7(anti-VLA-1), 5E9 (anti-transferrin receptor) conjugated with PE wereused to assess the activation and memory surface antigen phenotype ofthe Vα12.1⁺ -elevated CD8⁺ T cells in RA. The percentage expression ofeach of the receptors from the cells of the three patients wascalculated using the following formula: Vα12.1⁺ T cells: % LB3.1 (orother activation antigens) /Vα12⁺ (or CD8⁺) cells=(LB3.1⁺ cellsco-stained with anti-Vα12.1 (or anti-CD8) (2nd quadrant)/% T cells thatwere Vα12⁺ (or CD8+))×100.

The results revealed that, similar to most CD8⁺ T cells, Vα12.1 T cellsin RA patients expressed only IL-2R β chains. No significant amount offreshly isolated T lymphocytes expressed the high affinity IL-2R αβchains. Further, the transferrin receptor was not expressed on theVα12.1 elevated population of cells. However, CD45RO was expressed onthe majority of the Vα12.1 cells indicating a memory phenotype. Theseresults argue that the majority of Vα12.1 cells in circulation in theseRA patients were not acutely activated, but had previously beenactivated. In addition, 10 to 30% of Vα12.1⁺ T cells expressed VLA-1 andHLA-DR, suggesting ongoing activation for a minor subpopulation of thesecells.

EXAMPLE 6

This Example reveals that the expansion of Vα12.1 in CD8⁺ T cells of asubset of RA patients correlates with an increase in the occurrence ofthe HLA-DQW2 allele.

The MHC alleles present in eight patients from the Vα12.1-elevated groupwere compared to those with Vα12.1-normal group of RA patients.Serologic HLA typing for the RA patients was carried out at the Brigham& Women's HLA laboratory (Boston, Mass.) in accordance with establishedtechniques. The results of the HLA-typing for the 8 Vα12.1-elevatedpatients are illustrated in Table 1 of this Example.

                  TABLE 1                                                         ______________________________________                                        HLA Tissue Typing                                                             RA PATIENTS: CLASS I      CLASS II                                            ______________________________________                                        Patient #1   A.sub.--, B44,                                                                             :DR7,     DQw2                                      (6D6.sup.+ /CD8.sup.+ :43.%)*                                                              A32, B60, Cw3                                                                              :DR4,                                               Patient #2   A1, B8       :DR3,     DQw2                                      (6D6.sup.+ /CD8.sup.+ :29.%)                                                               A2, B.sub.--,                                                                              :DR4                                                Patient #3   A2, Bw52     :DR3,     DQw2                                      (6D6.sup.+ /CD8.sup.+ :22%)                                                                A11, B48     :DR10,                                              Patient #4   A2, B35, Cw4 :DR1,     DQw1                                      (6D6.sup.+ /CD8.sup.+ :27%)                                                                A11, Bw57, Cw3                                                                             :DR2,                                               Patient #5   A30, B14     :DR7,     DQw2                                      (6D6.sup.+ /CD8.sup.+ :26%)                                                                A23, B13     :DR1,     DQw1                                      Patient #6   A23, B27, Cw2                                                                              :DR4,     DQw2**                                    (6D6.sup.+ /CD8.sup.+ :20%)                                                                A24, Bw42,   :DR5,                                               Patient #7   A2, B7,      :DR3,     DQw2                                      (6D6.sup.+ /CD8.sup.+ :12%)                                                                A29, B44,    :DR7,     DQw4                                      Patient #8   A30, B7,     :DR3,     DQw2                                      (6D6.sup.+ /CD8.sup.+ :9%)                                                                 A11, B40,    :DR10,    DQw1                                      ______________________________________                                         *Percentage of CD8.sup.+  T cells that express Vα12.1 by surface        staining with mAb 6D6.                                                        **Confirmed with molecular probe.                                        

As shown in Table 1, no common class I was found to associatespecifically with this group of patients. The majority (>90%) of boththe Vα12.1-elevated and the Vα12.1-normal group of patients expressedHLA-DR1 and -DR4 alleles as expected for adult rheumatoid arthritispatients. Surprisingly, however, among the Vα12.1 elevated group ofpatients, the HLA-DQw2 allele was increased, as shown by thedetermination that 7 out of 8, or approximately 88% of the patients.

In general, the proportion of the DQw2 allele is approximately one inthree (depending upon ethnic background) and likewise, about 30% of theVα12.1 normal group of RA patients were DQw2-positive. The associationof HLA-DQw2 occurrence in the Vα12.1-elevated individuals was highlysignificant (chi square=11.4 p<0.001). The high frequency of HLA-DQw2 inthe Vα12.1 elevated group of patients implicates this class II moleculein the RA disease process. The expanded CD8⁺ T cells may be class II(DQw2)-restricted, or alternatively, are possibly reactive toDQw2-derived peptides recognized in the context of class I MHCmolecules.

These examples are provided for illustrative purposes and are notintended as a limitation on the spirit or scope of the presentinvention. Other embodiments will be apparent to persons skilled in theart.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (G) CELL TYPE: peripheral blood CD8+                                          T lymphocytes                                                                 (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: cDNA                                                             (B) CLONE: clone #1990, Microsoft Corp                                        (vii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: 14q                                                   (viii) FEATURE:                                                               (A) NAME/KEY: T cell receptor alpha                                           gene - JαAF211                                                          (B) LOCATION: 17 - 72                                                         (C) IDENTIFICATION METHOD: similarity with                                    known sequence                                                                (D) OTHER INFORMATION: Nucleotides 1- 12                                      comprise the 3'nucleotides of the rearranged                                  T cell receptor V region Vα12.1. Nucleotides                            13-16 are template independent insertions.                                    (x) PUBLICATION INFORMATION:                                                  (A ) AUTHORS: KLEIN et al.                                                    (B) TITLE: Diversity and structure of human T-                                cell receptor alpha chains.                                                   (C) JOURNAL: Proc. Natl. Acad. Sci.                                           (D) VOLUME: 84                                                                (F) PAGES: 6884                                                               (G) DATE: 1987                                                                (K) RELEVANT RESIDUES IN SEQ ID NO:1:                                         FROM 17 TO 72                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TGTG CTCTGAGTGATGGCTATGGTCAGAATTTT33                                          CYSALALEUSERASPGLYTYRGLYGLNASNPHE                                             GTCTTTGGTCCCGGAACCAGATTGTCCGTGCTG66                                           VALPHEGLYPR OGLYTHRARGLEUSERVALLEU                                            CCCTAT72                                                                      PROTYR                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (E) HAPLOTYPE:                                                                (G) CELL TYPE: peripheral blood CD8+T lymphocytes                             (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: cDNA                                                             (B) CLONE: clone #1990, Microsoft Corp                                         6.06, 6.07, 6.10, 6.11, 6.12, and 6.13.                                      (vii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: 14q                                                   (viii) FEATURE:                                                               (A) NAME/KEY: T cell receptor alpha gene JαA12                          (B) LOCATION: 17 to 78                                                        (C) IDENTIFICATION METHOD: similarity with known                              sequence                                                                      (D) OTHER INFORMATION: Nucleotides 1- 12 comprise                             the 3'nucleotides of the rearranged T cell                                     receptor V region Vα12.1. Nucleotides 13-16 are                        template independent insertions.                                              (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Koop et al.                                                      (B) TITLE: Nucleotide Sequence of the 3'                                      Terminal End of the TCR α/k locus.                                      (C) JOURNAL: Unpublished                                                      (D) VOLUME:                                                                   (F) PAGES:                                                                    (G) DATE: 1992                                                                (K) RELEVANT RESIDUES IN SEQ ID NO:2:                                          FROM 17 TO 78                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TGTGCTCTGAGTGATTATCAGGGCGGATCTGAA33                                           CYSALALEUSERASPTYRGLNGLYGLYSERGLU                                             AAGCTGGCTTTTGGA AAGGGAATGAAACTGACA66                                          LYSLEUVALPHEGLYLYSGLYMETLYSLEUTHR                                             GTAAACCCATAT78                                                                VALASNPROTYR                                                                  (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (G) CELL TYPE: peripheral blood CD8+T lymphocytes                              (vii) IMMEDIATE SOURCE:                                                      (A) LIBRARY: cDNA                                                             (B) CLONE: clone #1990, Microsoft Corp                                        1.09, 1.14, 1.15, 1.16, 1.17, 1.19, 1.25, 1.27,                               1.28, 1.31, 1.34, 1.37.                                                       (vii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: 14q                                                   (viii) FEATURE:                                                               (A) NAME/KEY: T cell receptor alpha gene JαA6                           (B) LOCATION: 13 - 72                                                         (C) IDENTIFICATION METHOD: similarity with known                               sequence                                                                     (D) OTHER INFORMATION: Nucleotides 1-11 comprise                              the 3'nucleotides of the rearranged T cell                                    receptor V region Vα12.1. Nucleotide 12 is a                            template independent nucleotide insertion.                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: TOYONAGO et al.                                                  (B) TITLE: Genes of the T-cell antigen receptors                              in normal and malignant T cells.                                              (C) JOURNAL: Ann. Rev. Immunol.                                               (D) VOLUME: 5                                                                 (F) PAGES: 585-620                                                            (G) DATE: 1987                                                                (K) RELEVANT RESIDUES IN SEQ ID NO:3:                                         FROM 13 TO 72                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TGTGCTCTGAGAGGGGGAGGTGCTGACGGACTC33                                           CYSA LALEUARGGLYGLYGLYALAASPGLYLEU                                            ACCTTTGGCAAAGGGACTCATCTAATCATCCAG66                                           THRPHEGLYLYSGLYTHRHISLEUILEILEGLN                                             CCCTAT 72                                                                     PROTYR                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           ( vi) ORIGINAL SOURCE:                                                        (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (G) CELL TYPE: peripheral blood CD8+T lymphocytes                             (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: cDNA                                                             (B) CLONE: clone #1990, Microsoft Corp                                        (vii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: 14q                                                   (viii) FEATURE:                                                               (A) NAME/KEY: T cell receptor alpha gene JαA6                           (B) LOCATION: 19 - 84                                                          (C) IDENTIFICATION METHOD: similarity with                                   known sequence                                                                (D) OTHER INFORMATION: Nucleotides 1- 14                                      comprise the 3'nucleotides of the rearranged                                  T cell receptor V region Vα12.1. Nucleotides                            15-18 are template independent insertions.                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: TOYONAGO et al.                                                  (B) TITLE: Genes of the T-cell antigen receptors                              in normal and malignant T cells.                                              (C) JOURNAL: Ann. Rev. Immunol.                                               (D) VOLUME: 5                                                                 (F) PAGES: 585-620                                                            (G) DATE: 1987                                                                (K) RELEVANT RESIDUES IN SEQ ID NO:3:                                         FROM 19 TO 84                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TGTGCTCTGAGTGAGCCTTATTCAGGAGGAGGT33                                            CYSALALEUSERGLUPROTYRSERGLYGLYGLY                                            GCTGACGGACTCACCTTTGGCAAAGGGACTCAT66                                           ALAASPGLYLEUTHRPHEGLYLYSGLYTHRHIS                                              CTAATCATCCAGCCCTAT84                                                         LEUILEILEGLNPROTYR                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (G) CELL TYPE: peripheral blood CD8+T lymphocytes                             (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: cDNA                                                             (B) CLONE: clone #1990, Microsoft Corp                                        (vii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: 14q                                                   (viii) FEATURE:                                                                (A) NAME/KEY: T cell receptor alpha gene JαA1                          (B) LOCATION: 17 - 72                                                         (C) IDENTIFICATION METHOD: similarity with                                    known sequence                                                                (D) OTHER INFORMATION: Nucleotides 1- 12 comprise                             the 3'nucleotides of the rearranged T cell                                    receptor V region Vα12.1. Nucleotides 13-16 are                         template independent insertions.                                              (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: KLEIN et al.                                                      (B) TITLE: Diversity and structure of human                                  T-cell receptor alpha chains.                                                 (C) JOURNAL: Proc. Natl. Acad. Sci.                                           (D) VOLUME: 84                                                                (F) PAGES: 6884                                                               (G) DATE: 1987                                                                (K) RELEVANT RESIDUES IN SEQ ID NO:1:                                         FROM 17 TO 72                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TGTGCTCTGAG TGACGGCTATGGTCAGAATTTT33                                          CYSALALEUSERASPGLYTYRGLYGLNASNPHE                                             GTCTTTGGTCCCGGAACCAGATTGTCCGTGCTG66                                           VALPHEGLYPROGLYTHR ARGLEUSERVALLEU                                            CCCTAT72                                                                      PROTYR                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (G) CELL TYPE: peripheral blood CD8+T lymphocytes                             (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: cDNA                                                             (B) CLONE: clone #1990, Microsoft Corp                                        17, 20, 21- 29, 31, 32, 34                                                     (vii) POSITION IN GENOME:                                                    (A) CHROMOSOME/SEGMENT: 14a                                                   (viii) FEATURE:                                                               (A) NAME/KEY: T cell receptor alpha gene JαA6                           (B) LOCATION: 10-70                                                           (C) IDENTIFICATION METHOD: similarity with                                    known sequence                                                                (D) OTHER INFORMATION: Nucleotides 1-11                                       comprise the 3'nucleotides of the rearranged                                  T cell receptor V region Vα12.1. Nucleotide 12                           is a template independent insertion.                                         (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: TOYONAGO et al.                                                  (B) TITLE: Genes of the T-cell antigen receptors                              in normal and malignant T cells.                                              (C) JOURNAL: Ann. Rev. Immunol.                                               (D) VOLUME: 5                                                                 (F) PAGES: 585-620                                                            (G) DATE: 1987                                                                (K) RELEVANT RESIDUES IN SEQ ID NO:3:                                         FROM 13 TO 72                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                      TGTGCTCTGAGAGGGGGAGGTGCTGACGGACTC33                                           CYSALALEUARGGLYGLYGLYALAASPGLYLEU                                             ACCTTTGGCAAAGGGACTCATCTAATCATCCAG 66                                          THRPHEGLYLYSGLYTHRHISLEUILEILEGLN                                             CCCTAT72                                                                      PROTYR                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (G) CELL TYPE: peripheral blood CD8+T lymphocytes                             (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: cDNA                                                              (B) CLONE: clone #1990, Microsoft Corp                                       (vii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: 14q                                                   (viii) FEATURE:                                                               (A) NAME/KEY: T cell receptor alpha gene JαA6                           (B) LOCATION: 19-84                                                           (C) IDENTIFICATION METHOD: similarity with                                    known sequence                                                                (D) OTHER INFORMATION: Nucleotides 1- 14                                      comprise the 3'nucleotides of the rearranged                                  T cell receptor V region Vα12.1. Nucleotides                            15-18 are template independent insertions.                                    (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: TOYONAGO et al.                                                  (B) TITLE: Genes of the T-cell antigen receptors                              in normal and malignant T cells.                                              (C) JOURNAL: Ann. Rev. Immunol.                                               (D) VOLUME: 5                                                                 (F) PAGES: 585-620                                                            (G) DATE: 1987                                                                (K) RELEVANT RESIDUES IN SEQ ID NO:3:                                          FROM 19 TO 84                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TGTGCTCTGAGTGAGCCTTATTCAGGAGGAGGT33                                           CYSALALEUSERGLUPROTYRSERGLYGLYGLY                                             GCTGACGGACTCACCTTTGG CAAAGGGACTCAT66                                          ALAASPGLYLEUTHRPHEGLYLYSGLYTHRHIS                                             CTAATCATCCAGCCCTAT84                                                          LEUILEILEGLNPROTYR                                                            (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 822 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: no                                                        (iv) ANTI-SENSE: no                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapien                                                     (D) DEVELOPMENTAL STAGE: adult                                                (G) CELL TYPE: Human T-cell leukemia line HBM-MLT                              (viii) FEATURE:                                                              (A) NAME/KEY: T cell receptor α- chain-encoding cDNA sequence           (B) LOCATION: 1-822                                                           (D) OTHER INFORMATION: Encoded amino acids -20 to -1 are                      predicted to be the leader sequence; encoded amino acids 1-95 are             predicted to be the variable region; encoded amino acids 96-111 are           predicted to be the joining region; encoded amino acids 112-228 are           predicted to be the constant region; encoded amino acids 229-248              are predicted to be the transmembrane region; and encoded amino               acids 249-253 are predicted to be the cytoplasmic region.                     (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS: Sim et al.                                                       (B) TITLE: Primary structure of human T-cell receptor α-chain           (C) JOURNAL: Nature                                                           (D) VOLUME: 312                                                               (F) PAGES: 771-775                                                            (G) DATE: 1984                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ATTTTTGCCAGCCTGTTGAGGGCAGTCATAGCC33                                           IlePh eAlaSerLeuLeuArgAlaValIleAla                                            20-10                                                                         TCCATCTGTGTTGTATCCAGCATGGCTCAGAAG66                                           SerIleCysValValS erSerMetAlaGlnLys                                            +1                                                                            GTAACTCAAGCGCAGACTGAAATTTCTGTGGTG99                                           ValThrGlnAlaGlnThrGluIleSerValVal                                             10                                                                             GAGAAGGAGGATGTGACCTTGGACTGTGTGTAT132                                         GluLysGluAspValThrLeuAspCysValTyr                                             20                                                                            GAAACCCGTGATACTACTTATTACTTATTCTGG165                                           GluThrArgAspThrThrTyrTyrLeuPheTrp                                            30                                                                            TACAAGCAACCACCAAGTGGAGAATTGGTTTTC198                                          TyrLysGlnProProSerGlyGluLeuVal Phe                                            40                                                                            CTTATTCGTCGGAACTCTTTTGATGAGCAAAAT231                                          LeuIleArgArgAsnSerPheAspGluGlnAsn                                             50                                                                            GAAATAAGTGGT CGGTATTCTTGGAACTTCCAG264                                         GluIleSerGlyArgTyrSerTrpAsnPheGln                                             60                                                                            AAATCCACCAGTTCCTTCAACTTCACCATCACA297                                          LysS erThrSerSerPheAsnPheThrIleThr                                            70                                                                            GCCTCACAAGTCGTGGACTCAGCAGTATACTTC330                                          AlaSerGlnValValAspSerAlaValTyrPhe                                              8090                                                                         TGTGCTCTGGACAGCAGTGCTTCCAAGATAATC363                                          CysAlaLeuAspSerSerAlaSerLysIleIle                                             100                                                                            TTTGGATCAGGGACCAGACTCAGCATCCGGCCA396                                         PheGlySerGlyThrArgLeuSerIleArgPro                                             110                                                                           AATATCCAGAACCCTGACCCTGCCGTGTACCAG429                                          AsnIleGlnAsnProAspProAlaValTyrGln                                             120                                                                           CTGAGAGACTCTAAATCCAGTGACAAGTCTGTC462                                          LeuArgAspSerLysSerSerAspLysS erVal                                            130                                                                           TGCCTATTCACCGATTTTGATTCTCAAACAAAT495                                          CysLeuPheThrAspPheAspSerGlnThrAsn                                             140                                                                           GTGTCACA AAGTAAGGATTCTGATGTGTATATC528                                         ValSerGlnSerLysAspSerAspValTyrIle                                             150                                                                           ACAGACAAAACTGTGCTAGACATGAGGTCTATG561                                           ThrAspLysThrValLeuAspMetArgSerMet                                            160                                                                           GACTTCAAGAGCAACAGTGCTGTGGCCTGGAGC594                                          AspPheLysSerAsnSerAlaValAlaTrpSer                                              170                                                                          AACAAATCTGACTTTGCATGTGCAAACGCCTTC627                                          AsnLysSerAspPheAlaCysAlaAsnAlaPhe                                             180                                                                           AACAACAGCATTATTCCAGA AGACACCTTCTTC660                                         AsnAsnSerIleIleProGluAspThrPhePhe                                             190200                                                                        CCCAGCCCAGAAAGTTCCTGTGATGTCAAG CTG693                                         ProSerProGluSerSerCysAspValLysLeu                                             210                                                                           GTCGAGAAAAGCTTTGAAACAGATACGAACCTA726                                          ValGluLysSerPheGluThrA spThrAsnLeu                                            220                                                                           AACTTTCAAAACCTGTCAGTGATTGGGTTCCGA759                                          AsnPheGlnAsnLeuSerValIleGlyPheArg                                             230                                                                           AT CCTCCTCCTGAAAGTGGCCGGGTTTAATCTG792                                         IleLeuLeuLeuLysValAlaGlyPheAsnLeu                                             240                                                                           CTCATGACGCTGCGGCTGTGGTCCAGCTGA822                                              LeuMetThrLeuArgLeuTrpSerSer***                                               250                                                                       

I claim:
 1. A method for detecting a subset of patients having adultrheumatoid arthritis, comprising the steps of:(A) contacting a samplecontaining T lymphocytes obtained from a subject suspected of havingrheumatoid arthritis with a Vα2.1-specific antibody, or fragment orderivative thereof reactive with an epitope of the gene product of theVα12.1 variable region of the alpha chain of the T cell receptor forantigen so as to form a detectable complex between the Vα12.1-specificantibody, fragment or derivative and the Vα12.1+ T lymphocytes; (B)contacting the sample with an anti-CD8 antibody that binds to a CD8 cellsurface antigen, so as to form a detectable complex between the CD8+ Tlymphocytes and the anti-CD8 antibody; (C) detecting the binding of theVα12.1 antibody, fragment, or derivative to the Vα12.1 gene product anddetecting the binding of the anti-CD8 antibody to the CD8 cell surfaceantigen and determining the number of CD8⁺ T lymphocytes expressingVα12.1; and (D) comparing the number of CD8⁺ T lymphocytes determined instep (C) expressing the Vα12.1 gene product with the number of CD8⁺ Tcells expressing the Vα12.1 gene product in a suitable base line sample,wherein an increased percentage of CD8+ T lymphocytes expressing theVα12.1 gene product (a Vα12.1 expansion) is diagnostic for rheumatoidarthritis (RA).
 2. A method according to claim 1, wherein the suitablebase line sample comprises a sample from a normal subject who does nothave RA.
 3. A method according to claim 2, wherein the subject is ahuman being.
 4. A method according to claim 3 wherein the sampleobtained from the subject is a sample of peripheral blood.
 5. A methodaccording to claim 4, wherein the Vα12.1 antibody, fragment, orderivative is conjugated to a label selected from the group consistingof fluorescent dyes, radioactive isotopes, enzymes, biotin, and metalions.
 6. A method according to claim 1, further comprising the step oftesting for clonality of the Vα12.1 expansion, wherein detecting aclonal or oligoclonal expansion is further diagnostic of RA.
 7. A methodaccording to claim 1 further comprising the step of HLA typing foroccurrence of a DQw2 allele, wherein detecting the occurrence of DQw2 isfurther diagnostic of RA.
 8. An immunoassay method for the detection ofa subset of human beings having adult rheumatoid arthritis whichcomprises:combining a body fluid sample containing DC8⁺ lymphocytes froma human being suspected of having adult rheumatoid arthritis with amonoclonal antibody or fragment or derivative thereof that bindsspecifically to an epitope of the Vα12.1 variable region of the T-cellantigen receptor; and determining whether expression of the Vα12.1 geneis increased on the CD8⁺ T lymphocytes of the human being as compared tothe CD8⁺ T lymphocytes of a normal human being that does not haverheumatoid arthritis, in which an expansion of Vα12.1 gene usage isdiagnostic for a subpopulation of human beings having rheumatoidarthritis.
 9. An immunoassay method according to claim 8, wherein theantibody comprises a murine monoclonal antibody.
 10. A method accordingto claim 8, wherein the antibody is conjugated to a label.
 11. A methodaccording to claim 8, further comprising the step of testing forclonality of the Vα12.1 expansion, wherein detecting a clonal oroligoclonal expansion of the Vα12.1 gene segment is further diagnosticof RA.
 12. A method according to claim 8, further comprising the step ofHLA typing for occurrence of a DQw2 allele, wherein detecting theoccurrence of DQw2 is further diagnostic of RA.
 13. A method accordingto claim 8, wherein the antibody, fragment or derivative is capable ofbinding to the same epitope of a T cell antigen receptor that isreactive with a monoclonal antibody secreted by a cell line having theATCC accession number HB
 10858. 14. A method for detecting a subset ofpatients having adult rheumatoid arthritis (RA), comprising the stepsof:(A) obtaining CD8+ T lymphocytes from a patient sample and isolatingnucleic acid from the CD8+ T lymphocytes; (B) determining the amount ofa Vα12.1 nucleic acid in the CD8+ T lymphocytes; and (C) comparing theamount of Vα12.1 nucleic acid in the CD8+ T lymphocytes in the patientsample with a control amount of Vα12.1 nucleic acid in a sample of CD8+T lymphocytes from a patient that does not have RA, wherein detecting anincreased amount of Vα12.1 nucleic acid (a Vα12.1 expansion) in thepatient sample compared to the amount in the control sample isdiagnostic for RA.
 15. A method according to claim 14, wherein thenucleic acid is DNA or mRNA.
 16. A method according to claim 14, whereinthe subject is a human being.
 17. A method according to claim 14,wherein the patient sample is from peripheral blood.
 18. A methodaccording to claim 14, further comprising the step of detecting theclonality of the Vα12.1 expansion, wherein detecting a clonal oroligoclonal expansion is further diagnostic of RA.
 19. A methodaccording to claim 14, further comprising the step of HLA typing foroccurrence of a DQw2 allele, wherein detecting of DQw2 is furtherdiagnostic of RA.